Method for pretreatment of microbial samples

ABSTRACT

Methods and devices are provided for pretreatment of a sample containing microbial cells. In some embodiments, the pretreatment of the sample is performed via the initial selective lysis, within a sample pretreatment vessel, of non-microbial cells (such as blood cells) and the subsequent centrifugal separation of the sample to remove the resulting debris and concentrate the microbial cells. An immiscible and dense cushioning liquid may be included for collecting the microbial cells adjacent to the liquid interface formed by the cushioning liquid upon centrifugation of the pretreatment vessel. After removal of a substantial quantity of the supernatant, resuspension of the collected microbial cells, and re-establishment of the cushioning liquid interface, at least a portion of the remaining suspension may be removed without substantially removing the cushioning liquid. One or more intermediate wash cycles may be performed prior to extraction of the remaining suspension, which provides a “pretreated” sample.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a National Phase application claiming the benefit ofthe international PCT Patent Application No. PCT/CA2013/000992, filed onNov. 26, 2013, in English, which claims priority to U.S. ProvisionalApplication No. 61/731,809, titled “APPARATUS AND METHOD FORPRE-TREATMENT OF MICROBIAL SAMPLES” and filed on Nov. 30, 2012, theentire contents of which is incorporated herein by reference, U.S.Provisional Application No. 61/750,242, titled “APPARATUS AND METHOD FORPRE-TREATMENT OF MICROBIAL SAMPLES” and filed on Jan. 8, 2013, theentire contents of which is incorporated herein by reference, and toU.S. patent application Ser. No. 13/833,872, titled “APPARATUS ANDMETHOD FOR PRE-TREATMENT OF MICROBIAL SAMPLES” and filed on Mar. 15,2013, the entire contents of which are incorporated herein by reference.

BACKGROUND

This disclosure relates to methods of detecting the presence and theidentity of microorganisms in a sample.

Emergence of drug resistant pathogens is a global healthcare crisis thatis forcing physicians to treat common infectious diseases with ever morepotent antibiotics. This is largely caused by the complexity and timerequired in identifying the offending bacteria, forcing physicians toprescribe empirically even with the knowledge of the high negativityrate amongst cultured specimens. The net result has been a significantincrease in emergence of resistant strains, higher treatment costs, andlonger recovery cycle due to an increase in side effect risks associatedwith taking broad spectrum and unnecessary antibiotics.

Broad spectrum antibiotics are commonly prescribed when treatingpatients that are exhibiting symptoms of sepsis or septic shock. Giventhe seriousness of these conditions, doctors will often prescribe one ormore broad-spectrum antibiotics right away and are not likely to changethe treatment regimen until the full effect of the drugs can be assessedor results from microbiology become available. For instance, thedocument “Surviving Sepsis Campaign Guideline” (SSCG) recommends atreatment protocol in which intravenous antibiotics, consisting of oneor more broad-spectrum agents against likely bacterial/fungal pathogens,should be started within the first hour of recognizing severe sepsis andseptic shock [R. P. Dellinger et al., Crit. Care Med 2008]. Thetreatment protocol states that the antimicrobial regimen is to bereassessed daily, and once the pathogen is known, as a matter of goodpractice, a more appropriate narrow-spectrum antimicrobial drug is to beadministered.

Unfortunately, current clinical bacteriology methods typically providepathogen identification information when it may be too late to impactpatient outcomes. This is due to the time lag of 2-3 days from specimencollection to reporting results of pathogen identification andsusceptibility testing. Causes for the time lag include the need totransport specimens to clinical laboratories staffed by expert clinicalbacteriologists and the time required for blood culture and subsequentcolony formation after plating the specimen on solid culture medium.Specimens that arrive at the clinical microbiology laboratory afternormal business hours are typically held overnight until staff arrivesthe next day. Once specimens are plated on solid agar on day 2 (after apositive blood culture has been obtained), an additional 8-12 hours areneeded for colonies to form. Plates are examined, colonies areenumerated, and appropriate colonies are selected for identification andsusceptibility testing. The process further requires analysis andinterpretation before reports are released, typically on day 3, whichmay be too slow to meaningfully impact antibiotic selection and patientoutcomes.

While many aspects of clinical microbiology laboratory workflow havebeen automated, clinical bacteriology remains highly labour-intensive.Many laboratories currently automate identification and susceptibilitytesting using either the Vitek (Biomerieux) or Phoenix(Becton-Dickenson) instruments. However, these systems, and newersystems based on mass spectroscopy, depend on selection of appropriatecolonies from overnight growth on agar plates by expert personnel.

Several nucleic acid amplification approaches for clinical bacteriologyhave recently been commercialized. However, most of these still requirea sample preparation process which includes a nucleic acid extractionand purification step that can take more than 3 hours, as part of thepre-analytical process for species identification. These processes arerequired for providing an inhibitor and contaminant free sample fornucleic acid assays. Consequently, despite their specificity andsensitivity, molecular methods such as PCR have not replaced the muchslower standard microbial culture-based techniques as the front linetest in the clinical microbiology laboratory, and the results fromclinical microbiology testing continue to be provided too late tosubstantially impact patient outcomes.

SUMMARY

Methods and devices are provided for pretreatment of a sample containingmicrobial cells. In some embodiments, the pretreatment of the sample isperformed via the initial selective lysis, within a sample pretreatmentvessel, of non-microbial cells (such as blood cells) and the subsequentcentrifugal separation of the sample to remove the resulting debris andconcentrate the microbial cells. An immiscible and dense cushioningliquid may be included for collecting the microbial cells adjacent tothe liquid interface formed by the cushioning liquid upon centrifugationof the pretreatment vessel. After removal of a substantial quantity ofthe supernatant, resuspension of the collected microbial cells, andre-establishment of the cushioning liquid interface, at least a portionof the remaining suspension may be removed without substantiallyremoving the cushioning liquid. One or more intermediate wash cycles maybe performed prior to extraction of the remaining suspension, whichprovides a “pretreated” sample.

The methods described herein may be useful in decreasing the time toresult of nucleic assays for the identification of microbial cells bytaking a different approach to the preparation of target nucleic acidsuspension. In some embodiments disclosed herein that involve a wholeblood sample, blood cells in the whole blood sample are selectivelylysed and their resulting debris are washed away while the microbialcells are kept intact and collected. The cell suspension is therebyrendered substantially free of molecular assay interferents andinhibitors. Subsequently, the microbial cells may be lysed with a methodthat does not require lytic reagents, and the lysate, which has beenrendered substantially free of molecular assay interferents andinhibitors, may be directly assayed, without nucleic acid extraction andpurification.

Accordingly, in one aspect, there is provided a method of extractingmicrobial cells from a whole blood sample, the method comprising:

adding a whole blood sample to a pretreatment vessel, the pretreatmentvessel comprising a pretreatment mixture, the pretreatment mixturecomprising a blood cell lysis reagent and a hydrophobic cushioningliquid having a density greater than that of the whole blood sample andthe blood cell lysis reagent;

agitating the contents of the pretreatment vessel;

centrifuging the pretreatment vessel such that the cushioning liquidforms a liquid interface below a supernatant, wherein microbial cellsfrom the whole blood sample are removed from suspension and collectedadjacent to the liquid interface;

withdrawing a substantial quantity of the supernatant without removingthe collected microbial cells;

agitating the contents of the pretreatment vessel and allowing thecushioning liquid to re-establish the liquid interface below asuspension containing the microbial cells; and

extracting a least a portion of the suspension without extracting asubstantial portion of the cushioning liquid, thereby obtaining anextracted suspension comprising microbial cells.

In another aspect, there is provided a pretreatment vessel forextracting microbial cells from a whole blood sample, comprising:

a vessel body defining an internal volume and an enclosure mechanism forsealing the vessel body, wherein the vessel body is configured for usein a centrifugation device;

the vessel body containing:

-   -   a pretreatment mixture comprising a blood cell lysis reagent,        and    -   a hydrophobic cushioning liquid having a density greater than        that of the whole blood sample and the blood cell lysis reagent,        wherein the cushioning liquid forms a liquid interface below a        supernatant upon centrifugation of said vessel body such that        microbial cells within a whole blood sample are removed from        suspension and collected adjacent to the liquid interface;

wherein the distal portion of said vessel body is conical in shape.

In another aspect, there is provided a method of extracting microbialcells from a liquid sample, the method comprising:

adding the liquid sample to a pretreatment vessel, the pretreatmentvessel comprising a pretreatment mixture, the pretreatment mixturecomprising a hydrophobic cushioning liquid having a density greater thanthat of the liquid sample;

agitating the contents of the pretreatment vessel;

centrifuging the pretreatment vessel such that the cushioning liquidforms a liquid interface below a supernatant, wherein microbial cellsfrom the liquid sample are removed from suspension and collectedadjacent to the liquid interface;

withdrawing a substantial quantity of the supernatant without removingthe collected microbial cells;

agitating the contents of the pretreatment vessel and allowing thecushioning liquid to re-establish the liquid interface below asuspension containing the microbial cells; and

extracting a least a portion of the suspension without extracting asubstantial portion of the cushioning liquid, thereby obtaining anextracted suspension comprising microbial cells.

In another aspect, there is provided a method of extracting microbialcells from a whole blood sample, the method comprising:

adding a whole blood sample to a pretreatment vessel, the pretreatmentvessel comprising a pretreatment mixture, the pretreatment mixturecomprising a blood cell lysis reagent;

agitating the contents of the pretreatment vessel;

centrifuging the pretreatment vessel such that the microbial cells fromthe whole blood sample are removed from suspension and collectedadjacent to the distal surface of the pretreatment vessel;

withdrawing a substantial quantity of the supernatant without removingthe collected microbial cells;

performing at least one series of wash steps, each series of wash stepscomprising:

-   -   adding a volume of wash buffer to the pretreatment vessel;    -   agitating the contents of the pretreatment vessel;    -   centrifuging the pretreatment vessel such that the microbial        cells are removed from suspension and collected adjacent to the        distal surface of the pretreatment vessel; and    -   withdrawing a substantial quantity of the supernatant without        extracting the collected microbial cells; and

agitating the contents of the pretreatment vessel to obtain a suspensioncontaining the microbial cells; and

extracting a least a portion of the suspension, thereby obtaining anextracted suspension comprising microbial cells.

In another aspect, there is provided a method of, the method comprising:

pretreating a sample containing microbial cells to obtain a suspensioncontaining the microbial cells;

lysing the microbial cells within the suspension to obtain a lysate; and

performing a multiplexed set of molecular assays on the lysate, themultiplexed set of molecular assays comprising:

-   -   at least one primary rRNA-based molecular assay for identifying        the kingdom of microbial cells present in the sample;    -   at least one secondary rRNA-based molecular assay for        identifying a Gram status of microbial cells present in the        sample; and    -   at least one tertiary rRNA-based molecular assay at the genus,        species level for each of fungi, Gram-positive bacteria, and        Gram negative bacteria;

wherein the at least one tertiary rRNA-based molecular assay for each ofeach of fungi, Gram-positive bacteria, and Gram negative bacteria aresuitable for the de-escalation of antimicrobial therapy based on theiroutcome.

In another aspect, there is provided an apparatus for extractingmicrobial cells from a sample, the apparatus comprising:

a motor;

a rotor driven by the motor, the rotor being configured to support apretreatment vessel such that a centrifugal force is applied to thecontents of the pretreatment vessel during rotation of said rotor,wherein the motor is configured to rotate the rotor at a speedsufficient for removing the microbial cells from suspension andcollecting the microbial cells near a distal surface of the pretreatmentvessel;

a rotating union comprising a rotating portion and a non-rotatingportion, wherein said rotating portion rotates in unison with saidrotor;

an aspirant tube extending from said rotating portion of said rotatingunion into the pretreatment vessel through a cap provided on thepretreatment vessel, said aspirant tube having a distal aspirant orificepositioned near the distal surface of the pretreatment vessel;

a dispensing tube extending from said rotating portion of said rotatingunion into the pretreatment vessel through said cap, said dispensingtube having a distal dispensing orifice positioned near the distalsurface of the pretreatment vessel;

a stationary entrance tube connected to said non-rotating portion ofsaid rotating union, such that said stationary entrance tube is in fluidcommunication with said dispensing tube during rotation of said rotor;

a stationary exit tube connected to said non-rotating portion of saidrotating union, such that said stationary exit tube is in fluidcommunication with said aspirant tube during rotation of said rotor; and

a pump mechanism in fluid communication with said stationary entrancetube and said stationary exit tube for dispensing and aspirating fluids.

A further understanding of the functional and advantageous aspects ofthe disclosure can be realized by reference to the following detaileddescription and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments will now be described, by way of example only, withreference to the drawings, in which:

FIGS. 1 (a) to (m) depict an example sample pretreatment vessel and itscontents at different stages during sample pretreatment.

FIGS. 2 (a) to (c) illustrate an alternative example embodiment forperforming pretreatment on a sample other than whole blood.

FIGS. 3 (a) and (b) illustrate another alternative example embodimentfor performing pretreatment on a sample other than whole blood.

FIGS. 4 (a) to (c) schematically illustrate three example pretreatmentvessels and their respective sedimentation zone in an angled centrifuge.

FIG. 4 (d) schematically illustrates centrifugal force acting on aparticle which has reached pretreatment vessel surface duringcentrifugation.

FIGS. 5(a) and 5(b) schematically depict example embodiment forautomating the sample pretreatment method, while FIG. 5(c) illustratesan example control and processing system.

FIGS. 6(a) to (f) illustrate alternative example embodiments forautomation of the sample pretreatment method.

FIG. 7(a) schematically depicts an example fluidic device for performingelectrical cell treatment, rRNA reverse transcription, PCR, and amplicondetection.

FIG. 7(b) schematically depicts the cross section A-A of electricalchamber 52 of FIG. 7(a) and shows the application of voltage pulses tothe electrical chamber.

FIG. 8 provides a flow chart illustrating an example method of detectingand identifying microbial cells in a whole blood sample.

FIG. 9(a) illustrates an example rapid identification panel forde-escalation of antibiotic therapy.

FIG. 9(b) illustrates example organism identification based on theresults from the rapid identification panel shown in FIG. 9(a), with acorresponding example antibiotic selection.

FIGS. 10(a) to (c) illustrate three example rapid identification panelsfor de-escalation of antibiotic therapy from broad-spectrum antibioticsto narrower spectrum antibiotics.

FIG. 11(a) shows the fluorescence signals of the amplicons in thesupernatant of each wash cycles, illustrating the quenching effect oflysed blood debris and how wash cycles can be employed to alleviatequenching.

FIGS. 11 (b) and (c) show the fluorescence signal versus PCR cyclenumber measured during real-time reverse transcription PCR (real timeRT-PCR) assays of the cell lysates in the supernatants of a samplepretreatment procedure in which wash cycles are employed withoutcentrifugation. FIG. 11 (b) shows the assay signals of cell lysates inthe supernatant of fourth wash cycle and FIG. 11 (c), the fifth washcycles, illustrating the dependence of the assay signal on the number ofwash steps and cell lysis method.

FIG. 11 (d) shows the fluorescence signal versus PCR cycle numbermeasured during real-time RT-PCR assays for cell lysates in thesupernatants of wash cycles using an example sample pretreatmentprocedure, illustrating the dependence of the assay signal on the numberof wash steps and electrical treatment.

FIGS. 12 (a) and (b) show the fluorescence signal versus PCR cyclenumber measured during real-time PCR assays, illustrating the dependenceof the assay signal on (a) the concentration of saponin, and (b) theconcentration of sodium polyanetholesulfonate (SPS).

FIG. 13 shows the fluorescence signal versus PCR cycle number measuredduring real-time PCR assays, illustrating the recovery of microbialcells from human whole blood with different types of anticoagulants.

FIGS. 14 (a) and (b) show the fluorescence signal versus PCR cyclenumber measured during real-time RT-PCR assays, illustrating thedependence of the assay signal on the volume of Fluorinert™ in (a) 2 mLand (b) 1.7 mL microcentrifuge tubes

FIG. 15 shows the fluorescence signal versus PCR cycle number measuredduring real-time RT-PCR assays, illustrating the dependence of the assaysignal on the pretreatment vessel surfaces.

FIG. 16 demonstrates the detection limit of Candida albicans in bloodsamples. The RT-PCR amplified product of the 18S rRNA fragment of C.albicans was visualized after resolving on agarose gel electrophoresis,showing the detection of 1 and 10 C. albicans cells.

FIG. 17 is a table showing the fluorescence intensity values obtainedfor the detection of Candida albicans in blood samples detected bymolecular beacon hybridization to RT-PCR amplified 18S rRNA.

FIG. 18 demonstrates detection limit of Streptococcus pneumoniae inblood samples. The RT-PCR amplified product of the 16S rRNA fragment ofS. pneumoniae was visualized after resolving on agarose gelelectrophoresis, showing the detection of 1 and 10 S. pneumoniae cells.

FIG. 19 is a table showing the fluorescence intensity values obtainedfor the detection of Streptococcus pneumoniae in blood samples detectedby molecular beacon hybridization to RT-PCR amplified 16S rRNA.

FIG. 20 demonstrates detection limit of Escherichia coli in bloodsamples. The RT-PCR amplified product of the 16S rRNA fragment of E.coli was visualized after resolving on agarose gel electrophoresis,showing the detection of 1 and 10 E. coli cells.

FIG. 21 is a table showing fluorescence intensity values for thedetection of Escherichia coli in blood samples detected by molecularbeacon hybridization to RT-PCR amplified 16S rRNA.

FIG. 22 is a table describing the microbial cells that were tested toshow the repeatability of the detection method.

FIG. 23 is a table describing the primers employed for RT-PCR assaysdescribed in FIGS. 25-27 which show the repeatability of the detectionmethod.

FIG. 24 shows the fluorescence signal measured during a typicalreal-time RT-PCR assay.

FIG. 25 shows the C_(T) values for the real-time RT-PCR detection of asingle cell of different Gram-positive bacterial species in bloodsamples.

FIG. 26 shows the C_(T) values for the real-time RT-PCR detection of asingle cell of different Gram-negative bacterial species in bloodsamples.

FIG. 27 shows the C_(T) values for the real-time RT-PCR detection of asingle cell of different fungal species in blood samples.

DETAILED DESCRIPTION

Various embodiments and aspects of the disclosure will be described withreference to details discussed below. The following description anddrawings are illustrative of the disclosure and are not to be construedas limiting the disclosure. Numerous specific details are described toprovide a thorough understanding of various embodiments of the presentdisclosure. However, in certain instances, well-known or conventionaldetails are not described in order to provide a concise discussion ofembodiments of the present disclosure. It should be understood that theorder of the steps of the methods disclosed herein is immaterial so longas the methods remain operable.

As used herein, the terms, “comprises” and “comprising” are to beconstrued as being inclusive and open ended, and not exclusive.Specifically, when used in the specification and claims, the terms,“comprises” and “comprising” and variations thereof mean the specifiedfeatures, steps or components are included. These terms are not to beinterpreted to exclude the presence of other features, steps orcomponents.

As used herein, the term “exemplary” means “serving as an example,instance, or illustration,” and should not be construed as preferred oradvantageous over other configurations disclosed herein.

As used herein, the terms “about” and “approximately”, when used inconjunction with ranges of dimensions of particles, compositions ofmixtures or other physical properties or characteristics, are meant tocover slight variations that may exist in the upper and lower limits ofthe ranges of dimensions so as to not exclude embodiments where onaverage most of the dimensions are satisfied but where statisticallydimensions may exist outside this region. It is not the intention toexclude embodiments such as these from the present disclosure. In someembodiments, the phrases “about” and “approximately” refer to ranges ofplus or minus 25% of the stated value.

In embodiments disclosed below, methods and devices are provided forsensitive detection and identification of microbial cells in a sample.In some embodiments, the pretreatment of the sample is performed via theinitial selective lysis of non-microbial cells (such as blood cells) andsubsequent centrifugation and optional wash cycles to remove theresulting debris and concentrate the microbial cells, where themicrobial cells are subsequently resuspended. The resulting microbialcell suspension is herein termed the “pretreated cell suspension”.

Sample Pretreatment Using a Cushioning Liquid

A method was disclosed by Dorn, for example, in U.S. Pat. Nos.5,070,014, 4,131,512, 4,212,948, 4,693,972, and 5,108,927, for lysing ablood sample and concentrating microbial pathogens for the purpose ofculturing and detection. Dorn teaches the use of a sample collectiontube that includes a blood lysis agent, containing saponin and sodiumpolyanetholesulfonate (SPS), and a cushioning liquid such as a highdensity liquid fluorinated hydrocarbon. A volume of whole blood is addedto the sample collection tube, and the saponin lyses blood cells in thesample. The cushioning liquid forms a distinct liquid layer at thebottom of the sample collection tube, and is provided to collectmicrobial cells at its liquid interface when the tube is centrifuged andretain the viability of the microbial cells which may otherwise bedamaged by impact or compaction forces in the resulting pellet. Afterremoval of the majority of the supernatant, Dorn teaches the removal ofall of the remaining liquid in the tube, including any residualsupernatant and all of the cushioning liquid.

The method of Dorn enjoyed commercial success prior to the advent ofautomated blood culture systems, as it was suitable for preparingmicrobial cells in a form that could be readily cultured. Unfortunately,the present inventors have found that the Dorn sample preparation methodis not suitable for many other applications, such as molecular assays,as the concentrated cell suspension obtained contains contaminants andinhibitory substances.

Many other known methods which employ blood lysing agents for thepreparation of cell suspensions followed by a microbial lysing step, orwhich use non-selective lysing methods to lyse both blood cells andmicrobial cells to prepare the sample, are also incompatible withdownstream molecular assays as they do not provide adequate removal ofcontaminants or inhibitory substances and therefore require the use ofnucleic acid extraction and purification methods prior to the molecularassay (Nierhaus, A., et al. “Comparison of three different commercialPCR assays for the detection of pathogens in critically ill sepsispatients.” Medizinische Klinik-Intensivmedizin and Notfallmedizin(2013): 1-8.).

Accordingly, in some embodiments of the present disclosure, devices andmethods are provided for the separation and concentration of microbialcells from a liquid sample, in which a cushioning liquid is optionallyemployed to collect microbial cells during a centrifugation step, butwithout substantially transferring the cushioning liquid when extractingthe pretreated sample. Furthermore, in some embodiments, one or morewash procedures are performed prior to extraction of the pretreatedsample in order to reduce the concentration of lysed blood cell debrisand other undesirable components and substances that may be present inthe pretreated sample. The reduction in concentration of thesesubstances in the pretreated sample may be beneficial in avoiding enzymeinhibition for molecular assays, reducing the presence of non-targetmacromolecules and preventing degradation of target macromolecules.

The retained microbial cells present in the pretreated sample may belysed and optionally subjected to treatment processes (example treatmentprocesses are described further below) for rendering the intracellularnucleic acid macromolecules (such as ribosomal RNA (rRNA) and DNA)available for subsequent molecular analysis. In some embodiments,subsequent amplification and detection steps may be employed for theidentification of the microbial cells. For example, the amplificationand detection steps may be performed directly on the pretreated andlysed sample without employing common nucleic acid extraction andpurification techniques. In particular, lysing techniques which do notemploy reagents that would otherwise interfere with amplification anddetection (for example, lysing techniques such as electrical, thermaland mechanical lysis) may be employed in this context.

In some embodiments, in which the sample is a blood sample containingblood cells (such as red blood cells and white blood cells) in additionto potentially containing microbial cells, the sample may be pretreatedin order to lyse the blood cells.

In embodiments of the present disclosure that involve performingmolecular assays after performing sample pretreatment, the compositionof the cell lysis reagent may be selected to be different from thatdisclosed by Dorn, because unlike the methods disclosed by Dorn thatrequired cell viability for subsequent analysis, cell viability may notbe required for molecular methods, and the threshold for acceptable cellinjury in the context of such molecular methods may be higher than thatof the Dorn protocol.

In some embodiments that involve the pretreatment of blood samples, theformulation of the blood lysis reagent is modified relative to thatdisclosed by Dorn in order to improve the efficiency of washing cycleswithout substantial loss of target nucleic acids. For example, animproved blood cell lysis reagent may be provided by selectingappropriate concentrations of saponin and SPS. Moreover, as describedbelow, other blood lysis reagents, such as, for example, Triton X-100(polyethylene glycol tert-octylphenyl ether), which in the context ofculture methods may inhibit cell growth, can be used in some of themethods disclosed herein. Examples of such blood lysis reagents aredescribed in detail below.

In some embodiments, aspects of the sample pretreatment method arecontrolled or prescribed such that: a high recovery of microbial cellsis obtained, such that a substantial percentage (e.g. greater than 10%,25%, 50%, 75%, or 95%) of the microbial cells present in the originalsample are transferred to the pretreated cell suspension; appreciableinjury is not inflicted on the microbial cells, such that the recoveredmicrobial cells preserve a substantial percentage (e.g. greater than10%, 25%, 50%, 75%, or 95%) of the target rRNA or mRNA or 100% of thetarget DNA; and the pretreated cell suspension liquid should besubstantially free of contaminants and substances that are detrimentalto or inhibit subsequent nucleic acid assays (e.g. in some applications,it has been found that such contaminants and/or interferents should havea concentration below approximately 0.1%). As described further below,various aspects of the methods described herein are suitable to beperformed as automated methods and devices.

FIGS. 1(a)-(m) illustrate an example implementation of a pretreatmentdevice and method according to one embodiment of the present disclosure,which may be employed for the pretreatment of whole blood samples, inwhich a cushioning liquid is employed. Referring to FIG. 1(a),pretreatment vessel 20 is provided containing a volume of blood celllysis reagent 22 and volume of cushioning liquid 21.

Pretreatment vessel 20 is a vessel suitable for centrifugation, such asa microcentrifuge tube. In some embodiments, the centrifuge tube has aconical bottom shape and a smooth inner surface, which minimisesadsorption or trapping of microbial cells during centrifugation. In someembodiments, pretreatment vessel 20 includes an injectable closuremember, such as pierceable stopper 25 (such as a rubber stopper), oranother suitable sealing mechanism that may be evacuated such that anappropriate volume of sample is drawn when the pierceable stopper ispierced by the needle of syringe 27.

In some embodiments, pretreatment vessel 20 is employed to obtain aselected volume of blood from a previously filled blood tube, such asanother blood collection tube. In some embodiments, pretreatment vessel20 may be employed as a sample collection device. For example,pretreatment vessel 20 may include a pierceable stopper that isconfigured for use with a Vacutainer®-type needle and holder device.

Cushioning liquid 21 is a high density and water immiscible liquid thatserves to form liquid surface 32 onto which the microorganisms settleduring centrifugation. As shown in FIG. 1(a), cushioning liquid 21 has adensity such that it settles at the bottom of pretreatment vessel 20under the influence of gravity. It will be understood that the term“high density”, as used herein with regard to cushioning liquid 21,means a density that is sufficiently high such that the target microbialcells will not substantially penetrate the cushioning liquid under theprevailing centrifugal force. The density of the cushioning liquid 21 istherefore chosen be greater than both the microbial cells and the otherliquids. The cushioning liquid is also immiscible in the other liquidsincluding, but not limited to, whole blood, blood cell lysis reagent 22,and a wash liquid (as described below) such that it remains a distinctliquid phase throughout the pretreatment process.

Blood cell lysis reagent 22 is an aqueous liquid that includes one ormore substances selected to lyse blood cells. In some embodiments, thecomposition of the blood cell lysis reagent is selected such that bloodcells are lysed without substantially affecting the integrity ofmicrobial cells. Examples of suitable compositions of blood cell lysisreagent 22 are provided below.

FIG. 1(b) illustrates the addition of a volume of blood sample 26 topretreatment vessel 20, where mixing of blood lysis reagent 22 with theblood sample 26 forms mixture 30 shown in FIG. 1(c). After providing asample to pretreatment vessel (e.g. through pierceable rubber stopper25), pretreatment vessel 20 may be agitated to produce further mixing ofthe sample with blood cell lysis reagent 22, as shown in FIG. 1(c). Forexample, pretreatment vessel 20 may be manually inverted one or moretimes to provide gentle mixing. In an alternative example, pretreatmentvessel 20 may be vortexed at a low speed. A non-limiting example of asuitable low vortexing speed is a speed near approximately 300 rpm for a4.9 mm orbit vortex mixer. An alternative mixing method can be employedin which the liquid is aspirated and dispensed for a number of cycles.

As stated above, the presence of blood cell lysis reagent 22 in mixture30 causes the selective lysis of blood cells. In one exampleimplementation, blood cell lysis reagent 22 may be an aqueous liquidincluding at least the following components: saponin, sodiumpolyanetholesulfonate (a sodium salt of polyanetholesulfonic acid, knownas SPS), and an antifoaming agent, such as poly (propylene glycol) (PPG,e.g. with a molecular weight of approximately 2000). In someembodiments, the saponin is purified saponin. Example methods forpurifying saponin are disclosed in U.S. Pat. No. 3,883,425, titled“Detoxification of Saponins”, which is incorporated herein in itsentirety. It has been found that without saponin purification blood celllysis is less efficient and a gel-like substance is formed uponcentrifugation.

In one example implementation, a composition of the blood cell lysisreagent, per approximately 1 mL of whole blood, may be provided asfollows: an aqueous solution having a volume of approximately 500 μLwith a concentration of approximately 75 mg/mL saponin (84510, Sigma),approximately 15 mg/mL sodium polyanetholesulfonate (SPS) (P2008, Sigma)and approximately 1% by volume of poly(propylene glycol) (PPG) MW 2000(202339, Sigma). Upon mixing the blood lysis reagent with the wholeblood sample the final concentrations of Saponin, SPS and PPG areapproximately 25 mg/ml, 5 mg/ml, and 0.3% respectively.

In one example implementation, the concentrations of saponin and SPS,upon mixing whole blood and the blood lysis reagent may be in the rangeof approximately 1.5 to 80 mg/mL and 0.5 to 20 mg/mL, respectively. Inanother example implementation, the concentrations of the saponin andSPS may be in the range of from approximately 10 to 30 mg/mL and 2.5 to10 mg/mL, respectively.

SPS is an anti-coagulant and anti-phagocytosis agent and is known toinhibit antimicrobial agents (Sullivan, N. M., Sutter, V. L., &Finegold, S. M. (1975). Practical aerobic membrane filtration bloodculture technique: development of procedure. Journal of clinicalmicrobiology, 1(1), 30-36.). The mechanism by which SPS assists in bloodcell lysis is not well understood. Without intended to be limited bytheory, it is believed that SPS may offer some level of protection tothe microorganisms during blood cell lysis, reduce the incidence ofentrapment of bacteria in cell debris, and/or reduce the amount ofcoagulated components which may otherwise be present in the sediment.

It is noted that SPS a known PCR inhibitor. At higher concentrations ofSPS, more washing cycles may be employed to remove excess SPS, forapplications in which a low concentration of SPS in the final extractedsample is desirable. It is also noted that U.S. Pat. No. 8,481,265describes the use of saponin with concentrations (the finalconcentration) in the range of 40-100 mg/ml as a lysis reagent, withoutthe use of SPS or a cushioning fluid. However, such an approach resultsin the pelleting of the microbial cells during centrifugation, anddifficulty in resuspending the pelleted microbial cells. In fact, in theexamples provided in U.S. Pat. No. 8,481,265, mechanical means wererequired for harvesting of the microorganism from the tube wall, whichis less amenable to automation.

The addition of PPG, an antifoaming agent, assists in maintaining asuitable viscosity of the mixture. It has been found that without theinclusion of PPG, the mixture may be thick and/or sticky.

In another example implementation, a composition of the blood cell lysisreagent, for lysing approximately 1 mL of whole blood, may be providedas follows: an aqueous solution having a volume of approximately 500 μL,with a concentration of approximately 1.5% by volume Triton X-100(X-100, Sigma), approximately 18 mg/mL sodium polyanetholesulfonate(SPS) (P2008, Sigma) and approximately 1% by volume poly(propyleneglycol) (PPG) MW 2000 (202339, Sigma) in a buffer pH ranging from 5 to11. Upon mixing the blood lysis reagent with the whole blood sample thefinal concentrations of Triton X-100, SPS and PPG are approximately0.5%, 6 mg/ml, and 0.3% respectively. In one example implementation, theconcentrations of Triton X-100 and SPS upon mixing the lysing reagentand whole blood sample may be in the range of approximately 0.5 to 1.5%and 5 to 10 mg/mL, respectively. In another example implementation, acomposition of the blood cell lysis reagent, for lysing approximately 1mL of whole blood, may be provided as follows: an aqueous solutionhaving a volume of approximately 1 mL, and with a concentration ofapproximately 1% Triton X-100 (X-100, Sigma) in a buffer pH ranging from9 to 11. In one example implementation, upon mixing the lysing reagentand whole blood sample the concentrations of Triton X-100 may be in therange of approximately 0.05 to 2.5%.

After forming mixture 30 and agitating pretreatment vessel 20,pretreatment vessel 20 is centrifuged, as illustrated in FIG. 1(d).Pretreatment vessel 20 is centrifuged at a suitable rate and for asuitable time to cause microbial cells in mixture 30 to pass out of thesuspension and collect at interface 32 between cushioning liquid 21 andsupernatant 33 as shown in FIG. 1(d).

It will be understood that the centrifugation time and speed required toeffect sedimentation of the microbial cells depends on the centrifugeradius, sample viscosity and density, and sedimentation path length, andmay be determined readily by those skilled in the art. For example, inone example embodiment, in which a blood volume of approximately 1 ml isinjected into a 2 ml pretreatment vessel (with a suitable quantity ofblood lysis reagents 22 and cushioning liquid 21 disclosed above) andplaced in a fixed angle centrifuge, a centrifugal force of approximately10000 g and a centrifugation time of approximately 1 minute issufficient to sediment microbial cells of interest. It will beunderstood that other combinations of time, speed, and sample volume maybe selected to achieve complete collection of the microbial cells at thecushioning surface 32.

As noted above, cushioning liquid 21 has a density greater than that ofmixture 30 and is immiscible with mixture 30 such that interface 32 isformed. This ensures that under centrifugal forces cushioning liquid 21rapidly moves to the sedimentation region 34 and displaces supernatant33, thereby forming a distinct fluid phase in the sedimentation region34 of pretreatment vessel 20. Cushioning liquid 21 prevents the loss ofmicrobial cells during the pretreatment process. Such a loss couldotherwise occur due to adsorption and trapping of microbial cells on thesurface of pretreatment vessel 20 within or near sedimentation region34, but is substantially prevented in the present embodiment due to thepresence of the liquid interface 32, where the microbial cells collect.Since the density of the cushioning liquid is greater than that of thecells the cushioning liquid prevents the cells from coming into closecontact with the vessel walls or entering cavities in the wall which mayotherwise trap said cells.

Fluorinated hydrocarbons, having molecular weight ranging from about 300to 850, are suitable as cushioning liquids. Examples are FC-40, FC-43,FC-70, and FC-77. For the examples provided in this disclosure,Fluorinert™ FC-40 (F9755, Sigma) was used. The cushioning liquid volumeshould be sufficiently large to provide an adequately largesedimentation surface so that all target precipitate is collected on itssurface. The cushioning liquid volume suitable for effective microbialcell recovery is dependent on a number of factors, including thepretreatment vessel geometry, pretreatment vessel surface condition,centrifuge angle, centrifugal force and target cell sedimentationcoefficient which are collectively termed centrifugal conditions.

One guiding principle for determining the volume of the cushioningliquid volume is that the surface of the pretreatment vessel wetted bythe cushioning liquid should at least envelop the region of thepretreatment vessel surface where the microbial cells would sediment inthe absence of the cushioning liquid. During centrifugation suspendedcells move radially and when they encounter the pretreatment vessel wallthey continue to move along the wall toward the extreme radial positionuntil the component of the centrifugal force in the direction of thepreferred movement is insufficient to overcome buoyancy and frictionalforces which oppose cell motion. In the absence of the cushioning liquidthe cells come to rest in a sedimentation region whose geometry reflectsthe centrifugal conditions. By providing cushioning liquid whichenvelops this region the cells will come into contact with thecushioning liquid either directly on its surface or on its periphery.

By way of illustration FIGS. 4 (a), (b) and (c) show three differentpretreatment vessel bottom angle geometries respectively in a 45 degreeangle centrifuge. In each case, the pretreatment vessel is shown inradial section view (along the rotor radius) as (i) and in a view fromthe rotor axis in a direction perpendicular to the pretreatment vesselaxis as (ii). In FIG. 4(a) the pretreatment vessel 120 has a bottom coneangle which is approximately 25 degrees and contains a fluid 121 inwhich the target microbial cells are initially suspended. The surface ofthis fluid is shown in FIG. 4(a) (i) and (ii) as 125. In the absence ofa cushioning liquid, after centrifugation for a sufficient duration atan appropriate speed, the cells will come to rest at or near theradially outermost portion 122 of the pretreatment vessel which in thiscase is at the intersection between the cylindrical and conical portionsof the pretreatment vessel. The sedimentation region 126 may extend adistance circumferentially on either side of the radially outermostpoint of the pretreatment vessel as shown in FIG. 4(a) (ii) due to thedecreasing net force in the direction of movement of sedimenting cellswhich are moving along the wall toward the extreme radial position.

When a cushioning liquid is initially present in the pretreatment vesseland centrifugation is performed in its presence, its surface 123 willapproximately form a vertical plane and the cushioning liquid will wetthe pretreatment vessel surface within the region 127 shownapproximately in FIG. 4(a) (ii). In order to ensure that sedimentingcells come into contact with the cushioning liquid before coming to reston the pretreatment vessel surface, the cushioning liquid should fullyenvelop the region 126 where sedimentation would have occurred in theabsence of the cushioning liquid.

Likewise, in a pretreatment vessel with a bottom cone angle of 45degrees which matches the centrifuge angle in this example, shown inFIG. 4(b) as 130, the cushioning liquid will form a surface 131 as shownin FIG. 4(b) (i). The sedimentation zone 132 in the absence of thecushioning liquid will generally be distributed along the cone as shownin FIG. 4(b) (ii). The surface region 133 of the pretreatment vesselwetted by the cushioning liquid should at least envelop thesedimentation zone 132.

When the pretreatment vessel bottom cone angle is greater than thecentrifuge angle as shown in FIG. 4(c) the sedimentation zone is at theapex of the cone and the cushioning liquid 143 should envelop thesedimentation zone 142.

It is evident from these examples that the minimum volume of cushioningliquid is dependent on the prevailing geometrical and sedimentationconditions. Swinging bucket or horizontally oriented centrifuges canlikewise be considered and will generally have a sedimentation region atthe apex of the pretreatment vessel, which should be enveloped by thecushioning liquid.

Other sedimentation vessels and centrifugal conditions can be similarlyconsidered to determine the minimum cushioning liquid volume.

It is noted that variability in the centrifugal conditions maynecessitate a cushioning liquid volume somewhat larger than the minimumdetermined as described above to ensure maximum recovery.

The sedimentation region on the surface of the pretreatment vessel whichprevails in the absence of the cushioning liquid, and which is wetted bythe cushioning liquid in accordance with the selected embodimentsdisclosed above, can be determined empirically. An example of anempirical method is the observation of the pellet of the sedimentedcells on the surface of the selected pretreatment vessel undercentrifugal conditions which are the same as those expected in thepretreatment process in the absence of the cushioning fluid. This may beassisted by fluorescent or other dyes applied to the cells to enhancethe optical detection of the sedimented cells. The volume of cushioningfluid suitable for enveloping this measured region could be determinedby observing the extent of the wetted region of the cushioning fluid onthe surface of the pretreatment vessel under the same centrifugalconditions and ensuring that this wetted region extends at least beyondthe previously observed sedimentation region (for example, by performingseveral experiments of cushioning liquid, and selecting the volume thatsufficiently wets the measured region). Alternatively, the recovery ofcells in the final suspension obtained by the methods described hereincan be measured by various means such as, for example, culturing andcolony enumeration, and a suitable volume of cushioning liquiddetermined empirically by selecting a volume that provides a suitable(e.g. maximum) cell recovery.

The volume of cushioning liquid can also or alternatively be estimatedby considering the interaction between the sedimenting particle and theinner surface of the pretreatment vessel during centrifugation. Duringcentrifugation, the particle whose density is greater than that of thesuspension fluid will move radially outward under the influence of thecentrifugal force vector {right arrow over (F)}_(c) of magnitude |{rightarrow over (F)}_(c)|=V_(p) (ρ_(p)−ρ_(m))ω²r until it reaches the surfaceof the pretreatment vessel. Here V_(p), ρ_(p), ρ_(m), ω, r represent thevolume of the particle, the density of the particle, the density of theparticle suspension medium, the angular velocity and the radial distancefrom the particle to the axis of rotation respectively.

Upon the particle 151 reaching this surface 150 where the unit outwardnormal vector to the surface is n as depicted in FIG. 4(d), the particlepath will be altered due to the presence of the pretreatment vesselwall. The particle trajectory will then be along the pretreatment vesselinner surface in the direction of the vector component of thecentrifugal force ({right arrow over (F)}_(ct)) in the tangent plane atthe particle position given by the relation {right arrow over(F)}_(ct)={right arrow over (F)}_(c)−({right arrow over (F)}_(c)·{rightarrow over (n)}){right arrow over (n)}.

Directly opposing this force will be fluid resistance whose magnitude iscommonly represented in the state of the art by the Stokes equationF_(s)=6πηrν, where η and ν are the fluid viscosity of the suspensionmedium and the velocity of the particle respectively, and a wallfrictional force F_(w), which, if of sufficient magnitude relative tothe other prevailing forces, should be included and determined for theparticular conditions of the pretreatment vessel surface, particle andsuspension fluid under consideration.

Thus the particle will reach a limiting or terminal velocity when forceequilibrium is achieved as represented by |{right arrow over(F)}_(ct)|=F_(s)+F_(w). A condition defining formation of a stablesediment may be defined by ν=0 or ν a sufficiently small value andthereby the above equations can be used to define a locus of points onthe pretreatment vessel surface at which the particles will effectivelycome to rest. This locus of points defines a sedimentation region on thepretreatment vessel inner surface which should be wetted by thecushioning fluid in accordance with the principles discussed above. As anumber of parameters in the above are subject to some amount ofvariation or uncertainty it is generally recommended to confirm theresults empirically.

It is also to be understood that the cushioning liquid surface shouldnot be so large as to cause loss of sedimented cells during aspirationof the supernatant during the centrifugal separation step.

In this respect, a consideration is the location of the sedimentationzone during centrifugation. Generally, some of the collected cells willsettle on the periphery of the cushioning liquid (referred to below as a“periphery collection region”) and some of such cells may remain on thepretreatment vessel wall even if the pretreatment vessel is reorientedfor aspiration. Since these cells are easily resuspended to the presenceof the cushioning liquid, and care should be taken not to disturb thisregion with convective disturbances in order to prevent loss of cells.Such convective disturbance may occur due to aspiration of thesupernatant.

In some embodiments the pretreatment vessel geometry and/or the volumeof cushioning fluid should be selected so that this periphery collectionregion remains distant from the aspiration device. Moreover, in someembodiments, the pretreatment vessel geometry and/or the volume ofcushioning fluid may be selected such that the periphery collectionregion remains submerged in the residual liquid during aspiration ofsupernatant.

For example, a pretreatment vessel (e.g. centrifuge tube) with a conicalor round (e.g. hemispherical) bottom (or a combination thereof, such asconical tapering side walls terminating in a hemispherical or otherwiseround bottom), may be employed to allow the sediment to remain in thebottom of the pretreatment vessel at a location that is distant from theaspiration device, and hence undisturbed while removing the supernatant.This becomes more important as the desired residual fluid volume isdecreased.

A suitable maximum volume of cushioning liquid is thereby related to anumber of factors, such as the desired residual fluid volume, thepretreatment vessel geometry, centrifuge angle and aspiration method.The distance between the aspiration tip and the cushioning fluid duringthe aspiration of the supernatant should be at least 1 mm and preferably3 mm to 5 mm or greater. An example volume of cushioning liquid 21 isapproximately 10 μL for a 2 mL pretreatment vessel in an anglecentrifuge operating at 6000 g. In another example embodiment, thevolume of cushioning liquid may range between approximately 5 μl and 15μl. In another example embodiment, the volume of cushioning liquid mayrange between approximately 2.5 μl and 100 μl.

After centrifugation, pretreatment vessel 20 may be re-oriented in aposition suitable for subsequent aspiration and dispensing operations,such as, for example, a vertical orientation as shown in FIG. 1(e), suchthat cushioning liquid 21 moves to the bottom of pretreatment vessel 20and remains there due to gravity. This step may be performedsufficiently slowly such that convective disturbances in the liquid areminimised such that most or all of the microbial cells are preventedfrom being resuspended. The sedimented microbial cells may be depositedon the surface of the vessel proximal to the location of thesedimentation region or some cells may move with the cushioning liquidas it relocates under gravity.

The prevention of resuspension of cells enables the removal of most ofthe supernatant, as shown in FIG. 1(f), such that only a small volume ofresidual supernatant is left behind containing the retained microbialcells. For example, a syringe may be employed to remove a substantialportion of the supernatant. In order to achieve concentration of theretained microbial cells and to remove substances that could interferewith downstream processes, it is beneficial to remove as much of thesupernatant as possible while leaving behind a sufficient volume ofsupernatant such that removal of the microbial cells in the aspirant issubstantially avoided. Such an embodiment is effective in reducing thetransfer of the blood cell debris and lysis reagents to the subsequentwashing stages, and to the final pretreated sample. It is to beunderstood that in some embodiments, step (e) and step (f) may beperformed with different orientations and positions of the pretreatmentvessel and aspiration device. For example, in step (e) the pretreatmentvessel may remain in the orientation of step (d) but be gently rotated180 degrees in its holder such that the sedimentation region is nowlocated at the bottom of the angled pretreatment vessel. This will allowaspiration of a greater amount of supernatant while leaving thesedimentation region undisturbed.

In one embodiment, the amount of the supernatant that is removed isbetween 80% and 95% of the total amount of supernatant. In an exampleimplementation employing the quantities according to those described inthe above example, a suitable aspirant volume is approximately 1.35 mL.

As shown in FIG. 1(g)-(k), one or more washing cycles may be optionallyperformed to purify the supernatant and to reduce the concentration ofblood cell debris and blood cell lysis reagent present in the pretreatedsample. Referring to FIG. 1(g), a volume of the washing liquid 35 may beadded to sample pretreatment vessel 20. After addition of washing liquid35, the solution is mixed to resuspend debris that may have sedimentedor adsorbed to the vessel wall during centrifugation. This may beaccomplished by vortexing as shown in FIG. 1(h). Pretreatment vessel 20is subsequently centrifuged and re-oriented, and a substantial portionof the supernatant is removed, as shown in FIGS. 1(i) to (k), similar tothe manner described for FIGS. 1(d) to (f).

In one example implementation, washing liquid 35 may be any low ionicstrength aqueous medium such as salt solutions or buffers. The choice oflower ionic strength enables using the microbial cell lysate, followingelectrical or mechanical cell lysis, for molecular assays withoutrequiring an ion balance step.

As noted below, the microbial cells are suspended in residual washingliquid 37 when the pretreated sample is extracted from pretreatmentvessel 20. Accordingly it may be beneficial for the washing liquid 35 tohave a composition suitable for downstream processing of the sample. Inone example implementation, washing liquid 35 may include one or morereagents or media for preserving the viability of the retained microbialcells. For example, washing liquid 35 may include monovalent saltsolution such as potassium chloride or sodium chloride. In anotherexample, the washing liquid is a buffer such as phosphate buffer pH 7.4,Tris buffer pH 8.0. In another example, the washing liquid is a bufferthat is compatible with downstream nucleic acid amplification reactions.In embodiments in which electrical lysis is performed subsequent toextraction of the pretreated sample (as described below), the ionicstrength of the washing liquid should be selected such that it iscompatible with working parameters of the electrical lysis method. Forexample, for the case of lysing microbial cells via electrical method,the ionic strength of washing liquid 35 may be between approximately 0.1and 1 mM.

The number of wash cycles employed during sample pretreatment may dependon various factors, such as the desired level of dilution of theresidual supernatant 36. For example, in an embodiment in which thesample is a blood sample and the blood lysis reagent includes SPS, itmay be useful to dilute the supernatant 36 such that the residualconcentration of SPS is less than approximately 0.01 μg/mL.

In one example implementation, the wash procedure is not performed priorto extraction of the processed sample. This embodiment is particularlysuited when the intended nucleic acid assay uses inhibitor-resistant PCRreagents, examples of which have been described by A. T. Hall et al(“Evaluation of Inhibitor-Resistant Real-Time PCR Methods forDiagnostics in Clinical and Environmental Samples.” PloS one 8.9 (2013):e73845.). It is known that high level of haemoglobin left in thesuspension quenches the fluorescence signal that typically is employedfor monitoring real-time PCR assays. It is understood that theinhibitor-resistant PCR reagents often do not offer the rapid cyclingtime, therefore wash cycles may be desirable when fast detection time isrequired.

In another embodiment, one wash cycle may be employed. In otherembodiments, two or more wash cycles may be employed. In one example,based on the example parameters disclosed above, four wash cycles werefound to provide effective removal of contaminants and inhibitorysubstances from the sample for downstream processing.

After performing the (optional) washing cycles, pretreatment vessel 20may be agitated as is shown in FIG. 1(l), such that the retainedmicrobial cells are resuspended in the residual supernatant 37. Forexample, pretreatment vessel 20 may be vortexed for approximately 5 to20 seconds at a low speed, as described above.

After agitation, cushioning liquid 21 is allowed to settle at the bottomof pretreatment vessel 20, as shown in FIG. 1(m), such that pretreatmentvessel 20 includes a residual suspension of the retained microbial cellsabove the cushioning liquid 21. This residual suspension may then beremoved to obtain the extracted sample, referred to as the pretreatedsample 38.

In one embodiment, a substantial volume of the residual suspension isremoved, such that little or none of cushioning liquid 21 is removed.Such an embodiment is beneficial in removing most of the retainedmicrobial cells, while avoiding contamination by cushioning liquid 21.For example, the controlled removal of a selected volume of the residualsuspension may be achieved via the insertion of a syringe withcalibrated length, which may be inserted through pierceable rubberstopper 25, leaving cushioning liquid 21 behind.

In some embodiments, the portion of the suspension that is removed doesnot include cushioning liquid. In another embodiment, the portion of thesuspension that is removed is substantially free of the cushioningliquid, such that the presence of any cushioning liquid would not havean impact on the performance of a subsequent assay. In anotherembodiment, all of the residual suspension is removed, which may furtherinclude a small fraction of cushioning liquid 21, such as, for example,<1%, <2%, <5%, or <10%. In some embodiments, an acceptable amount ofcushioning liquid that can be withdrawn may be determined based onknowledge of an acceptable concentration of the cushioning liquid in asubsequent assay (i.e. such that assay performance is not degraded). Insome embodiments, a portion of the cushioning liquid may be removed,where the volume of cushioning liquid that is removed is less thanamount that would reduce the performance of a subsequent assay below apreselected level. For example, in the case of a downstream assayinvolving real-time PCR, experiments may be performed to determine thevolume of cushioning liquid that can be removed without reducing thecycle number below a pre-selected threshold for a given standard analyteconcentration (e.g. a performance deficit of less than one cycle). Inother assay formats, other suitable measures can be employed, such asthreshold values associated with the limit of detection and the signalintensity.

In yet another embodiment, in which one or more optional wash cycles areperformed, both the residual suspension and a substantial portion ofcushioning liquid may be removed, and the cushioning liquid may beexternally separated from the residual suspension. For example, in oneembodiment, both the residual suspension and a substantial portion ofthe cushioning liquid may be removed by an aspiration vessel such as apipette tip. In this case the cushioning liquid may be separated fromthe residual suspension by allowing it to settle adjacent to a lowerorifice of the aspiration vessel (e.g. the pipette tip) and subsequentlybe dispensed from the orifice of the aspiration vessel or allowed todrip from the orifice of the aspiration vessel without loss of theresidual suspension. In another example embodiment, external filtrationmay be employed to separate the suspension from the cushioning liquid.

Although the preceding example embodiments relate to the processing ofwhole blood as a sample matrix, it is to be understood that the methodsdisclosed herein may be adapted to the pretreatment of a wide variety ofspecimens. Suitable specimens include, but are not limited to, urine,sputum, cerebral spinal fluid, swabbed tissue samples, vaginal samples,and other sample types of biological origin, and non-biological samplesthat may contain microbial cells. A sample may be provided by processinga solid or partially solid sample in order to produce a liquid sample(e.g. using a process such as homogenization). Examples of other sampletypes include other liquid samples that may contain microbial cells,such as environmental water samples, liquid food samples, andhomogenized food samples. The initial sample may be combined with areagent, buffer, or other medium prior to introduction into thepretreatment vessel.

FIGS. 2 and 3 illustrate two additional example embodiments for thepretreatment of a biological sample. Both FIG. 2 and FIG. 3 show theinitial steps of alternative sample pretreatment embodiments, where theremaining steps are preformed according to the remaining steps (byFigure sub-letter) shown in FIG. 1.

In FIG. 2, an embodiment is shown in which sample pretreatment vessel 20includes a pretreatment buffer, reagent or liquid 100 that may or maynot include a blood cell lysis reagent. For example, liquid 100 may beuseful in stabilizing a sample other than a whole blood sample, such assputum. Sample is added in step (b) and pretreatment vessel 20 isagitated in step (c), with the remainder of the steps performed asdescribed above in relation to FIG. 1(a)-(m).

Similarly, FIG. 3 shows an example embodiment in which samplepretreatment vessel 20 initially includes cushioning liquid 21, to whichsample, or a sample combined with a buffer, reagent or other liquid, isadded in step (b). The remainder of the steps are performed as describedabove in relation to FIG. 1(a)-(m). Such an embodiment may be useful forthe pretreatment of a urine sample, for example. Alternatively, thepretreatment of a urine sample may be performed according to a methodsimilar to that disclosed in FIG. 2, where liquid 100 includes a bloodcell lysis reagent for the lysis of blood cells that may be present inthe urine sample.

Sample Pre-Treatment without Cushion Liquid

The preceding methods and devices employ the use of a pre-treatmentvessel with a cushioning liquid. The cushioning liquid prevents a lossin cell recovery (or assay signal in the case of downstream molecularassays) that would otherwise occur (in the absence of the cushioningliquid) due to the adhesion of microbial cells to the walls of thepretreatment vessel.

For example, as illustrated in FIG. 14(a), and as described in Example4.4 below, the inclusion of 10 μL of Fluorinert™ in a siliconizedplastic microcentrifuge tube produced a gain of four PCR cycles relativeto a pretreatment vessel without a cushioning liquid. This resultsuggests that the inherently rough or adsorbent surfaces of plasticmicrocentrifuge tubes, even when treated with siliconization, result ina substantial reduction in recovery when a cushioning liquid is notused. Therefore, in the case of plastic pre-treatment vessels, both theuse of a cushioning liquid and the hydrophobic treatment of the vesselsurface appear to play significant roles in enabling a high recovery ofmicrobial cells in the extracted suspension.

In other implementations, however, the pretreatment vessel may be formedfrom a material having a smoother surface than the plasticmicrocentrifuge tubes described above, and the smoother surface mayallow for suitable or acceptable recoveries in the absence of thecushioning liquid. For example, it has been shown by Dorn, in U.S. Pat.No. 4,164,449, that a glass centrifuge tube, having a siliconizedsurface, may be suitable for pretreatment in the absence of a cushioningliquid, while enabling high recovery values. It is noted, however, thatthe recovery values reported by Dorn were based on inoculation of a 1000microbial cells, and that the recovery may be considerably lower forlower inoculation numbers. It is also noted that the Dorn methodinvolved the withdrawal of the major portion of the supernatant after asingle centrifugation step, followed by the resuspension of the retainedmicrobial cells in the residual supernatant. Such a method would beincompatible with downstream molecular assays due to the interferentspresent within the residual supernatant that is used to form thesuspension.

Accordingly, in one embodiment, a pretreatment method may be performedaccording to the embodiments described above, but without employing thecushioning liquid, provided that the pretreatment vessel has a smoothinner surface having a hydrophobic surface treatment (such as a glasscentrifuge tube having a siliconized surface). Unlike the methoddisclosed by Dorn, which was suitable for subsequently growth analysis,the method according to the present embodiment employs one or moreintermediate wash steps prior to the withdrawal of the final suspensionof microbial cells. The one or more intermediate was steps may beperformed according to any of the washing methods described in thepresent disclosure. The one or more intermediate wash steps result in ahigher purity microbial suspension, with a substantially reducedconcentration of interferents that would otherwise inhibit or prohibitsubsequent (downstream) molecular assays. Such purity was not importantto the method of Dorn, in which the suspension was employed for growthpurposes, as opposed to for molecular assays that are susceptible tointerferents present in the supernatant of a lysed blood samplefollowing an initial centrifugation step. Accordingly, the presentembodiment, in which one or more intermediate wash steps are employed ina pretreatment method absent of a cushioning liquid, can be employed toprovide a suspension having a high recovery of microbial cells whilealso having a dramatically lowered concentration of interferents, suchthat the suspension is suitable for subsequent molecular assays.

In some embodiments, a pretreatment method may be performed withoutusing a cushioning liquid, where a pretreatment vessel having a smoothhydrophobically-treated inner surface is employed (such as a glasscentrifuge tube having a siliconized surface), and where the distalportion of the pretreatment vessel has a conical or rounded (e.g.hemispherical) shape that is suitable for extraction of the supernatant(after centrifugation) while leaving a residual volume of less thanapproximately 100 ul, or less than approximately 50 ul. Such anembodiment results in a concentrated microbial cell suspension (purifiedby optional wash steps), that is suitable for performing molecularmethods. This is in stark contrast to the method of Dorn, in which thefinal suspension was employed for subsequent growth analysis, withoutrequiring or employing a low-volume concentrated suspension.

Automated Methods of Sample Pre-Treatment

In some embodiments, the sample pretreatment processes disclosed above(with or without a cushioning liquid), or variations thereof, may beautomated to reduce the operator involvement and to improve cellcollection and washing effectiveness. For example, a centrifuge that isconfigured with the ability to perform automated wash steps may beemployed for this purpose.

FIGS. 5(a)-(c) illustrate an example implementation of an automateddevice for use in sample pretreatment, including a centrifugationmechanism that is integrated with a washing mechanism. FIG. 5(a) shows adiametrical cross-sectional view of a centrifuge with a closed aspirantpath. The centrifuge is powered by a motor capable of achieving speedsand centrifugal forces described in the aforementioned embodiments.

Rotor 40 contains receptacles for a plurality of pretreatment tubes 41.A dispensing tube 42 and an aspirant tube 43 are inserted into thepretreatment tube 41 by piercing the pierceable top cap of thepretreatment tube. Dispensing tube 42 and aspirant tube 43 may beprovided according to a number of different configurations, such asconcentrically, or in a parallel/adjacent configuration as shown in FIG.5 The dispensing and aspiration tubes, or the plurality of dispensingtubes and aspiration tubes in the case of multiple pretreatment tubesfor simultaneous pretreatment, terminate at a manifold 44 which isfluidically connected to a rotating union 45. The rotating union 45 isprovided to allow fluidic connection during rotor rotation of thedispensing and aspiration paths to entrance and exit tubes 46 and 47respectively which are stationary. Such rotating unions are availablecommercially and may be designed for rotational speeds disclosed herein.

Entrance tube 46 and exit tube 47 may be connected to a dispensing fluidsource and waste reservoir respectively, each of which are provided witha dispensing mechanism (such as a peristaltic pump or a syringe pump)for independently dispensing and aspirating fluids. The vortexing,dispensing, and aspiration actions may be controlled independently bythe device in accordance with a pre-programmed treatment protocol,following one or more of the steps outlined in FIG. 1(a)-(m), in anautomated fashion, instead of a manual fashion. Vortexing may beaccomplished, for example, by varying rotational speed, rotationalstop/start cycles, rapid alternating rotational direction cycles, andother suitable motions of rotor 40. Alternatively a vibratory mechanismmay be applied to the rotor at rest or located on the rotor to apply theforces necessary to resuspend sediment and mix the fluids as required bythe pretreatment process described. For example the centrifuge tube maybe directly vortexed by a motorised orbiting mechanism. Alternatively apiezo driven vibrator or a motorised vibrator comprising a motor and aneccentric mass on its rotating shaft may be employed in this respect.Following sedimentation steps, aspiration may be performed while rotor40 rotates at a speed for which the cushioning liquid and cells are heldin the sedimentation region with the aspiration tube so positioned as toprevent disturbance of the sedimented microbial cells and avoidaspiration of the cushioning liquid or the microbial cells. With properplacement of the aspiration tube, a small residual fluid volume may beachieved which in turn may increase the wash efficiency. For example aresidual volume of 100 ul, 50 ul, or 25 ul may be achieved. For exampleFIG. 5(b) illustrates an aspiration tube placement 48 and residual fluidvolume 49 which may be achieved without disturbing the sedimented cellsand the cushioning fluid when aspiration occurs during rotor rotation.

The pretreatment protocol may be performed with the automated centrifugewasher in the following manner. With reference to FIG. 1, the sampleinsertion steps (b) may be performed manually and the pretreatment tubesso prepared are placed in the centrifuge washer. The dispensing andaspiration tubes are inserted into the pretreatment tubes as describedabove and the manifold connections made. The centrifuge washer ispre-programmed and operated in accordance with the desired parametersfor the subsequent series of vortexing, centrifugation, aspiration anddispensing steps analogous to the steps of FIG. 1 (c) to (l) in order toperform the sample pretreatment and washing actions. It is to beunderstood that some or all of the steps shown in FIGS. 1(c)-(l) may beautomated without re-orienting the pretreatment tube. The pretreatmenttubes are then removed from the centrifuge washer and the aspirationstep of FIG. 1(m) is performed manually.

As shown in FIG. 5(c), centrifuge mechanism 270, and liquid dispensingmechanism 280, may be connected to, or connectable to, a control andprocessing unit 225 for controlling the operation of the liquiddispensing and centrifuge mechanisms according to the presentlydisclosed sample pretreatment methods. Control and processing unit 225may include computer or processing hardware such as one or moreprocessors 230 (for example, a CPU/microprocessor), bus 232, memory 235,which may include random access memory (RAM) and/or read only memory(ROM), one or more internal storage devices 240 (e.g. a hard disk drive,compact disk drive or internal flash memory), a power supply 245, onemore communications interfaces 250, external storage 255, a display 260and various input/output devices and/or interfaces 255.

Although only one of each component is illustrated in FIG. 5(c), anynumber of each component can be included in the control and processingunit 225. For example, a computer typically contains a number ofdifferent data storage media. Furthermore, although bus 232 is depictedas a single connection between all of the components, it will beappreciated that the bus 232 may represent one or more circuits, devicesor communication channels which link two or more of the components. Forexample, in personal computers, bus 232 often includes or is amotherboard.

In one embodiment, control and processing unit 225 may be, or include, ageneral purpose computer or any other hardware equivalents. Control andprocessing unit 225 may also be implemented as one or more physicaldevices that are coupled to processor 230 through one of morecommunications channels or interfaces. For example, control andprocessing unit 225 can be implemented using application specificintegrated circuits (ASICs). Alternatively, control and processing unit225 can be implemented as a combination of hardware and software, wherethe software is loaded into the processor from the memory or over anetwork connection.

Control and processing unit 225 may be programmed with a set ofinstructions which when executed in the processor causes the system toperform one or more methods described in the disclosure. Control andprocessing unit 225 may include many more or less components than thoseshown.

FIGS. 6(a)-(e) illustrate alternative devices which allow automation ofsome steps of the pretreatment process. FIG. 6(a) portrays anelutriation device 300 which is operated in accordance with theprinciples of counterflow centrifugal elutriation. The elutriationdevice consists of an inlet tube 301, a conical collection chamber 302opening in the direction of fluid flow, and an exit cone 303 and outlettube 304. The elutriation device is placed in a centrifuge rotor androtated such that the centrifugal force 305 or a sufficient componentthereof is directed along the axis of the elutriation device. The fluidsuspension is passed through the device flowing from the inlet tube 301,through the collection chamber 302, and exiting through the outlet tube304. While passing through the collection chamber the particles aresubjected to centrifugal forces which oppose the flow direction suchthat particles with sedimentation coefficients less than a predeterminedmagnitude are prevented from flowing out of the collection chamber asthe fluid flows outward through the chamber outlet 304. The collectionzone 306 within the collection chamber is dependent on the centrifugalforce and flow velocity both of which vary with position and can bepredetermined by choosing appropriate chamber dimensions and centrifugeoperating speed.

In the context of the current disclosure the initial pretreatment stepsof FIG. 1(a) to (c) are completed in a separate pretreatment vessel andthe pretreatment fluid/sample mixture is passed through the elutriationdevice as described above. The cells thereby remain suspended in thecollection chamber while the full volume of the pretreatment fluidmixture is passed through the chamber. A further volume of wash bufferis passed through the chamber so as to displace the pretreatment fluidand produce a clean cell suspension thus eliminating any contaminantsthat may be detrimental to subsequent sample processing. In thisembodiment the presence of the cushion liquid is not required as cellsare held in suspension throughout the centrifugal process and thereforeloss of cells due to entrapment or adsorption on the chamber surfacewill not occur. The final pretreated cell suspension is obtained bylowering the centrifuge speed and/or increasing the flow rate therebyallowing the cell suspension to exit through the outlet tube 304 to apretreated sample collection device. Alternatively the cell suspensionmay be removed by reversing the flow and removing the fluid from thechamber via the inlet tube 301 either during or after stoppingcentrifugal rotation.

Since it is also generally necessary to concentrate the cells in asmaller fluid volume than the original sample, the elutriation chambershould be of an appropriate size to allow extraction of the suspendedcells into the desired pretreated sample volume. Thus an elutriationchamber volume may be similar in size to the final sample. For example,the pretreatment fluid/sample volume used in examples herein isapproximately 1.5 ml and a preferred final clean cell suspension volumeis approximately 50 μl for some applications. For a typical elutriationchamber geometry with a volume of approximately 50 μl and typicalbenchtop centrifuge parameters the elutriation flow rate is limited toapproximately 1 μl/second for which the total elutriation time amountsto processing times of approximately 30 minutes. This processing timecan be substantially reduced by initially performing steps (a) to (f)followed by steps (l) and (m) of FIG. 1 to obtain a concentratedpretreatment fluid/sample volume and performing the wash steps in thecurrent elutriation device with the reduced volume.

An alternative pretreatment device 310 is shown in FIG. 6(b) which issimilar in function to the elutriation device of FIG. 6(a) but differsin some aspects. In this case the elutriation chamber has a closedbottom 311 and the inlet tube 312 is inserted through the outlet tube313 such that the incoming fluid is dispensed close to but at apredetermined distance away from the bottom of the tube. The outgoingfluid flows from the annular outlet 314 created by the inlet and outlettubes. The operation of this device is similar to that of theelutriation device described above but its design allows access to boththe chamber inlet and outlet from one end of the device. A relatedembodiment is shown in FIG. 6(c) in which the elutriation chamber 320 isintegral to a centrifuge tube 321 and the elutriation chamber outlettube 322 exits into an isolated waste chamber 324. A similar embodimentcan be envisaged for the flow-through embodiment of FIG. 6(a). The wastechamber prevents contamination of external waste flow paths and retainsall fluid waste within the device which is advantageous for safeoperation and disposal of potentially hazardous sample fluids. For thisembodiment it is preferred to remove the cell suspension by drawing thefluid from the chamber through the inlet tube 323 in the reversedirection to the fluid flow during operation. The outlet tube 322extends beyond the surface of the waste fluid under the conditionsprevailing when washing is complete and the clean cell suspension isremoved from the elutriation chamber.

A further embodiment is shown in FIGS. 6(d) and 6(e), which extends theconcept of FIG. 6(c) to a two part device consisting of a first part 330which is the centrifuge tube and the second part 331 which includes theelutriation exit cone 332, inlet tube 333, outlet tube 334 and wastechamber 335 as shown in FIG. 6(d). The main body of the second part 331has a cylindrical outer surface 336 and is insertable into thecylindrical portion 340 of the first part and may be pushed into saidfirst part until the bottom portion 336 of the second part engages withthe bottom portion 341 of first part as illustrated in FIG. 6(e). Theextremity of the bottom portion of the second part is so formed that itseats tightly with the bottom portion of the first part such that a sealis obtained between the first and second parts thus forming an isolatedchamber 345 with inlet tube 333 and outlet tube 334. A deformableportion or material such as a rubber gasket may be provided at theextremity 337 of the second part to provide an adequate seal.

The pretreatment fluid/sample mixture 342 containing the cushioningfluid is initially dispensed into the first part 330 and subjected tocentrifugation such that the microbial cells sediment on the surface andperiphery of the cushioning fluid 346 at the bottom apex of the tube.Optionally the first part 330 contains a volume of blood lysis reagentand cushioning liquid as described previously and the sample isinserted, followed by mixing so as to produce a mixture and effect lysisof the blood cells, and is subsequently subjected to centrifugation.

Following sedimentation of the microbial cells the second part 331 isinserted into and pushed to the bottom of the first part 330sufficiently slowly to allow the supernatant so displaced to flowthrough the outlet tube and into the waste chamber resulting in theengaged position of the parts as shown in FIG. 6(e). In this way, alarge portion of the supernatant 347 is separated from the chamber 345and a washing step may proceed within the smaller chamber volume. Hencethe washing function can proceed by rotating the engaged tube assemblywhile flowing wash fluid through the inlet tube into the chamber. Cellswhich were sedimented on the cushioning liquid at the bottom of thechamber may be disturbed by the incoming flow but will be retained inthe chamber due to centrifugal forces which exceed flow induced forceson the cells due to the controlled flow rate. The wash fluid willdisplace pretreatment fluid and non-sedimenting particles to produce aclean fluid in the chamber.

In order to improve the washing efficiency the fluid in the chamber maybe mixed by vibration or vortexing intermittently in order to resuspendparticulate that may attach to the chamber wall and to dilute fluidzones of relative stagnation. Furthermore the density of the wash fluidmay be increased to a level exceeding that of the pretreatmentfluid/sample mixture by, for example, the addition of trehalose toprovide a more effective displacement of pretreatment fluid/samplemixture from the chamber. FIG. 6(f) illustrates an alternate embodimentfor which the second part 331 is inserted into a first part 350 prior tooperation of the device. Optionally the inlet tube 333 may be a separatedevice which is inserted through a pierceable membrane 351 at the top ofthe tube. The inlet tube 333 may optionally also be used to insert thesample or the pretreatment fluid/sample mixture into the tube.

Integration of Sample Pre-Treatment with Downstream Molecular Processing

In some embodiments, the preceding embodiments may be employed for theinitial extraction and concentration of microbial cells, followed bydownstream methods for identifying microbial cells using reversetranscription-PCR (RT-PCR); the reverse transcription of rRNA intocomplementary DNA (cDNA), with subsequent amplification and detection ofthe cDNA by PCR. The amplification of cDNA may optionally be performedsimultaneously, or serially, with the amplification of genomic DNA,where the cDNA and the gDNA are employed for identifying differentgenotypic taxa of the microbial cells. The gDNA may be obtained from thesame lysate as the rRNA.

Microbial cells that may be identified according to embodiments of thedisclosure include bacteria and fungi. In some embodiments, rRNA fromthe microbial cells is released, and a reverse transcription step isemployed to obtain cDNA, where the cDNA is representative of a firstgenotypic taxa level, and where the cDNA, if present, is subsequentlyamplified and detected in order to identify the first genotypic taxalevel of the microbial cells. Simultaneously or sequentially,amplification and detection of gDNA released from the microbial cells isemployed to identify a second genotypic taxa of the microbial cells of asecond level, where the second level is of a lower level than the firstlevel. In another embodiment, mRNA may be used as a target for themicrobial identification, and a reverse transcription step is employedto obtain cDNA and where the cDNA, if present, is subsequently amplifiedand detected in order to identify the second genotypic taxa level of themicrobial cells. This embodiment may be preferred when the informationon the viability of the microorganism is desired. As it is known in theart, mRNA content of microorganisms drop following the cell death.

An example module for performing electrical treatment, reversetranscription of rRNA, PCR, and amplicon detection is schematicallypresented in FIG. 7(a). Electrical channel 52 includes an inlet port 51through which fluid sample and other fluids may be introduced.Electrical channel 52 also includes outlet port 53, which is in fluidcommunication with downstream thermal chamber 54, where the reversetranscription of rRNA and PCR may be performed. Flow along electricalchannel 52 is provided by a pressure differential between inlet 51 andoutlet 53 ports.

The device may include additional fluid features, such as valves foropening and closing ports 51 and 53. For example, valves 57 and 58 mayoptionally be provided at the inlet 51 and outlet ports 53 of electricalchannel 52, and valve 59 is optionally provided at the outlet of thermalchamber 54. Valves 57, 58 and 59 may employ any suitable valvingmechanism compatible with a microfluidic channel, including, but notlimited to, pinch valves, ball valves, disc valves, plug valves. Valves57 and 58 may be provided to assist in controlling evaporation of theliquid during electrical lysing and treatment and to control theexposure of the fluid to electrical field and thermal effects. Forexample, valves 57 and 58 may be useful in ensuring that a sufficientlyhigh temperature is achieved for sufficient and/or efficient electricallysis (for example, some organisms, such as fungi, may require highertemperatures and/or temperature rates of change for efficient lysis).

Referring now to FIG. 7(b), cross section A-A of electrical chamber 52of FIG. 7(a) is shown along with a circuit for applying voltage pulsesto the electrical chamber. Electrical treatment of the processed samplemay be performed, for example, according to methods disclosed inco-pending PCT Application Number PCT/CA2012/000698, titled “METHODS ANDDEVICES FOR ELECTRICAL SAMPLE PREPARATION” and filed on Jul. 25, 2012,which is incorporated herein by reference in its entirety. The deviceincludes a thin channel defined on one side by the top plate 61, upperelectrode 63 and on the opposite side by base plate 62 and lowerelectrode 64. The upper and lower portions are separated by a thinspacer, in which material is removed to form the channel cavity. Thespacer may be made from a dielectric material, which may be slightlydeformable under an applied clamping pressure, or which is bonded to theupper and lower surfaces of the channel cavity. The spacer thus definesthe side walls of the channel, provides the fluid seal, and electricallyinsulates the top and bottom electrodes from each other.

The lower electrode 64 and upper electrode 63 are electrically isolatedfrom the base and top plates (substrates) by lower and upperelectrically insulating layers. In accordance with thermal requirements,thermal insulating layers may also be provided which may be separatefrom or be suitable selections of the electrical insulating material.The channel has dimensions H×W×L which, in one example implementation,used for the tests in the following examples, was 0.2×6.4×15 mm³. Thetwo electrodes 63 and 64 are intended for inducing an electric fieldacross the channel resulting in the establishment of an ionic current.

As per the methods disclosed in PCT Application NumberPCT/CA2012/000698, the application of suitable amplitude modulatedelectric pulse train by the external voltage source 68 on the twoelectrodes, establishes an electric field in the electric chamber. Theelectric field results in ionic current and Joule heating of the liquid.As the duration of the electric pulse train and, consequently, theaccompanying Joule heating is brief, this mechanism of heating is knownas flash heating. The coupled effect of the electric field acting on thecells and the flash heating of the liquid causes the microbial cells tolyse and intracellular molecules, such as proteins and nucleic acids, tobe released from the cell. The lysis process, irreversibly permeabilizesthe microbial cell membrane and readily supports molecular exchange inand out of the cell.

The thermal properties of the channel are dependent on many differentchannel parameters. For example, the channel conductivity and heatcapacity can be controlled according to the geometry and/or thickness ofthe metal electrode. Most electrodes will have a high thermalconductivity, but the thermal properties of the channel can be tailoredby selecting an appropriate electrode thickness to provide a suitableheat capacity and an appropriate (e.g. thermally insulating) substrateupon which the electrodes are supported.

Accordingly, one or more of the channel electrodes may be provided as ametal foil or coating having a high thermal conductivity and/or a highheat capacity relative to the total volume of fluid in the channel topromote rapid cooling after electrical treatment, while at the same timeproviding a sufficiently small heat capacity such that the flash heatingcan produce a rapid temperature rise within the channel during theapplication of the voltage pulses (for example, a temperature risegreater than approximately 250 degrees Celsius per second).

Generally speaking, the following ranges may be employed for electricalprocessing of microbial cells. The electric field strength within thechannel that is produced by the application of the voltage pulses mayrange between approximately 200 V/cm<E<50 kV/cm, depending on the typeof cell that is to be processed and the degree of electrical processingthat is desired. A range of approximately 2 kV/cm<E<30 kV/cm may bepreferable for lysis of microorganisms and using the lysate forperforming diagnostic tests on the released nucleic acids.

According to different example implementations, the pulse width ofindividual voltage pulses may range between approximately 1 μs<t_(p)<10ms, depending on the type of cell that is to be processed and the degreeof electrical processing that is desired. A range of approximatelyt_(p)<1 ms may be preferable for avoiding the electrical breakdown ofthe dielectric coating in the case of blocking electrodes, minimizingthe accumulation of the electrochemical products in the case ofnon-blocking electrodes. A range of approximately t_(p)>10 μs ispreferred for lowering the high frequency demands of drivingelectronics.

According to other example implementations, the time duration over whichthe voltage pulses are applied may be less than about 5 s, depending onthe type of cell that is to be processed and the degree of electricalprocessing that is desired. In some cases, such as to minimize the heatinduced degradation of target macromolecules and decrease the powerdemands of the driving electronics, an effective time duration forelectrical processing may be less than about 100 ms.

According to other example implementations, the ionic strength of thecell containing liquid may range from approximately 0.1 mM<I<100 mM,depending on the ionic composition of the initial sample that is to beprocessed and the degree of electrical processing that is desired. Insome cases, when filtering is used and fluid exchange is allowed, a moresuitable range for the ionic strength may be from approximately 0.1mM<I<10 mM, or 0.2 mM<I<1 mM.

According to other example implementations, the peak temperature of theliquid within the channel during the application of voltage pulses mayrange from approximately 30° C.<T_(p)<250° C., depending on the type ofcell that is to be processed and the degree of electrical processingthat is desired. In some applications, such as lysis of microorganismsand using the lysate for performing diagnostic tests on the releasednucleic acids, it may be preferable for the temperature range to liewithin approximately 80° C.<T_(p)<200° C.

The heating rate of the liquid for the electrical processing may begreater than approximately 250° C./s, depending on the type of cell thatis to be processed and the degree of electrical processing that isdesired. In some cases, such as lysis of Gram positive bacteria, fungi,and spores, a suitable range may include rates greater than about 2000°C./s.

The cooldown time of the liquid following electrical treatment may beless than approximately 1 s, depending on the thermal sensitivity of thetarget macromolecule. In some cases, such as when the targetmacromolecule is particularly sensitive, a preferred range may includetimes below about 100 ms.

The macromolecular content of the cell may undergo a transformation inthe period between turning on of the electric field and cooling down ofthe liquid. This process, referred to herein as “E-treatment”, mayrender nucleic acids, such as rRNA and gDNA, more accessible to enzymes,thus improving the efficiencies of the ensuing nucleic acid reversetranscription and amplification processes. Moreover, E-treatment may beeffective in inactivating most of enzymes that are released frommicrobial cells or have been left in the channel from residual bloodcell debris. Thus, the deleterious inhibitory effects of such enzymes inthe subsequent processes may be minimized. Other inhibitors that hinderstranscriptase and/or polymerase activity may be also rendered lesseffective or benign through E-treatment.

Referring again to FIG. 7(a), according to one example implementation,thermal chamber 54 is provided for performing reverse transcription ofrRNA followed by the PCR. (e.g. one-step RT-PCR). The RT-PCR master mixmay be introduced in liquid form or may be present in the chamber in dryformat prior to the introduction of the sample. After the introductionof the treated sample into thermal chamber 54 and mixing with the RT-PCRmaster mix, the resulting mixture is incubated to perform reversetranscription of rRNA and thermally cycled to perform PCR. In oneexample embodiment, the heating and cooling operations may be providedby placing the device on a Peltier device. In other embodiments, heatingmay be provided by electrical resistance contact heaters, radiantheating, or convection heaters and cooling may be provided bycirculating fluids, forced air flow, and other suitable methods.

According to this example implementation, the nucleic acid sequences ofthe rRNA are reverse-transcribed to complementary DNA (cDNA). Thereagent for reverse transcription typically contains reversetranscription enzyme, deoxy nucleotide triphosphate mixture (dNTPs), anappropriate buffer with magnesium or manganese cofactor, and optionallyRNase inhibitor. The reverse transcription can be any RNA-dependent DNApolymerase enzyme known in the prior art such as Moloney Murine LeukemiaVirus (M-MuLV) reverse transcription, Avian Myeloblastosis Virus (AMV)reverse transcription and Thermus thermophilus (Tth) DNA polymerases.

The primers can be non-specific random primers or gene-specific primers.In some embodiments, nucleic acid sequences of no more than 500 baseswill be reverse transcribed to cDNA using gene-specific primers. Thespecific primers can be primer sets or degenerate primers. One or moretarget nucleic acid sequences of one or more microorganisms will bereverse transcribed at the same time.

In one embodiment, three sets of primers, respectively targetingGram-positive, Gram-negative, and fungal rRNA, are simultaneously used(multiplexed). Reverse transcription takes place at an appropriatetemperature depending on the enzyme and on the primer annealingtemperature (for example, for no more than 10 minutes). In addition, thereaction mixture may contain ingredients for performing PCR, includingTaq DNA polymerase, deoxy nucleotide triphosphate mixture (dNTPs).Optionally, RNase inhibitors, Taq DNA polymerase antibody for hot-start,adjuvants to inhibit PCR inhibitors (example; bovine serum albumin) orto enhance PCR performance (example; betaine) may be included.

The PCR cycles may involve an initial incubation at 94-98° C. for 2-5minutes, to inactivate the reverse transcription enzyme from the firststage, and to activate DNA polymerase for PCR, followed by a 2-step or3-step thermal cycling. The thermal chamber may contain one or morespecific primer sets or degenerate primers. In addition to the primers,one or more components of PCR reagent may also be provided in thechamber in a dry format, such as freeze-dried reagent, or ambienttemperature dried reagent employing appropriate stabilizers.

In the example implementation illustrated in FIG. 7(a), in order toperform the multiplexed detection of amplicons produced during PCR, thecontents of thermal chamber 54 are transferred into plurality of wells56 via channels 55. Each well may be pre-filled, optionally in dryformat, with appropriate nucleic acid detection reagents. Accordingly,spatially multiplexed detection may be performed. In one embodiment,spatially multiplexed detection may be performed using molecular beaconsfor signal generation. In other embodiments, multiplexed detection maybe performed by melting curve analysis of the amplicons stained with adouble-stranded DNA dye, or employing fluorophores with differentemission wavelengths.

In one embodiment, the detection is performed using molecular beacons.Molecular beacons have a stem-and-loop structure, with the loop portionbeing complementary to a single-stranded DNA target while the stem isformed by 6 to 8 nucleotides from two complementary arm sequences. Afluorophore (such as, but not limited to, fluorescein (6-FAM) isattached to the end of one arm, while a quencher (such as but notlimited to dabcyl succinimidul ester) is attached to the other arm. Inthe absence of the specific target, the molecular beacon remains in a“dark” state. In the presence of the specific target, the fluorophoreand quencher are separated and the fluorescence emission, resulting fromexcitation by light with appropriate spectrum, can be detected by anoptical system. In the preferred embodiment, the excitation anddetection are performed using epi-fluorescence microscopic objectivethat may utilize an LED light source. In some embodiments, the molecularbeacon may be stored in wells 56 in a solution or in dry format.

According to one example implementation, a primer pair to detect allbacterial 16S rRNA genes may be CGGCTAACTCCGTGCCAGCAG (SEQ. ID. 1) andATCTCTACGCATTTCACCGCTACAC (SEQ. ID. 2), which targets a hypervariableregion of target bacterial species (nucleotides 504 to 697 usingEscherichia coli O104:H4 str. 2011C-3493 as a reference). A primer pairto detect target fungal species could be AGGGGGAGGTAGTGACAATAAAT (SEQ.ID. 3) and CAAAGTTCAACTACGAGCTT (SEQ. ID. 4), which targets a variableregion of eukaryotic 18S rRNA (nucleotides 436 to 622 using Candidaalbicans AB013586 as a reference). The molecular beacons cansubsequently be used to identify specific nucleotide patterns withinthis amplified region to distinguish between the pathogens of interest.For example, a molecular beacon designed to detect target Gram-negativespecies could be 6-FAM-5′-CCGAGCGGTGCAAGCGTTAATCGGAATTACTGGGCGCTCGG-3′-DABCYL (SEQ. ID. 5), which targets a region ofthe 16S rRNA gene (nucleotides 541-569 using Escherichia coli O104:H4str. 2011C-3493 as a reference) conserved in all Gram-negative bacteria.Alternatively, a molecular beacon designed to detect target fungalpathogens could be6-FAM-5′-CCGAGCTCTGGTGCCAGCAGCCGCGGTAATTCGCTCGG-3′-DABCYL (SEQ. ID. 6),which targets a region of the 18S rRNA gene (nucleotides 539 to 564using Candida albicans AB013586 as a reference) conserved in all fungi.Using this strategy, target bacterial and fungal blood pathogens can beidentified using a small number of primer pairs and several molecularbeacons. The molecular beacons could ultimately be multiplexed, allowingone to identify multiple bacterial and/or fungal pathogens in a singlechamber.

In another example embodiment, microbial cells may be identified bytwo-step RT-PCR. Thermal chamber 54 in FIG. 7(a) is provided forperforming reverse transcription of rRNA. The RT-PCR master mix may beintroduced in liquid form or may be present in the chamber in dry formatprior to the introduction of the sample. After the introduction of thetreated sample into thermal chamber 54 and mixing with the RT-PCR mastermix, the resulting mixture is incubated to perform reverse transcriptionof rRNA. In order to perform the multiplexed detection of the target,the contents of thermal chamber 54, containing cDNA and the master mixrequired for PCR are transferred into plurality of wells 56 via channels55. Each well may be pre-filled, optionally in dry format, withappropriate specific primer sets. The nucleic acid detection reagentssuch as double-stranded DNA dyes may be provided in the RT-PCR mastermix and spatially multiplexed detection may be performed by real-timePCR in detection wells 56.

In another example embodiment, thermal chamber 54 in FIG. 7(a) may beprovided for introduction of RT-PCR master mix which may be introducedin liquid form or may be present in the chamber in dry format prior tothe introduction of the sample. After the introduction of the treatedsample into thermal chamber 54 and mixing with the RT-PCR master mix,the resulting mixture is incubated to ensure proper mixing of themixture. In this example embodiment, microbial cells may be identifiedby real-time one-step RT-PCR. In order to perform the multiplexeddetection of the target, the contents of thermal chamber 54, containingthe cell lysate and the master mix required for RT-PCR are transferredinto plurality of wells 56 via channels 55. Each well may be pre-filled,optionally in dry format, with appropriate specific primer sets. Thenucleic acid detection reagents such as double-stranded DNA dyes may beprovided in the RT-PCR master mix and spatially multiplexed detectionmay be performed by real-time one-step RT-PCR. Referring now to FIG. 8,a flow chart is provided that illustrates an example method ofperforming microbial classification and/or identification according toone embodiment (e.g. using RT-PCR followed by signal detection usingmolecular beacon). In step 10, sample pretreatment is performedaccording to the preceding embodiments. The result is a cell suspensionin a liquid with desired composition that is appropriate for thesubsequent sample treatment and nucleic acid amplification steps. Thesample pretreatment step may also be useful for substantially reducingor eliminating the presence of DNA fragments from dead microbes that canbe introduced while taking sample from infected patients.

Electrical processing, by which the microbial cells are lysed and theintracellular rRNA and genomic DNA are rendered accessible for externalenzyme action, is performed in step 11, as described above. Electricaltreatment in this step may also inactivate or reduce the effect ofenzymes or factors inhibitory to subsequent amplification and detection.In step 12, the pretreated and lysed sample is mixed with an appropriatemaster mix in the thermal chamber and targeted regions of the rRNA arereverse transcribed into corresponding cDNA. The master mix containscomponents for performing cycles of PCR amplification on the cDNA orspecific regions of gDNA, as shown in step 13.

In the method and embodiments disclosed, the three preceding steps,namely 11, 12 and 13, may be performed in fluidic sequence in a singledevice in order to prevent or reduce losses in the number of targetmolecules. Such losses are associated with liquid exchanges andmovements in commonly used methods for sample extraction and/orpreparation. Accordingly, this aspect of the present method, along withthe efficient sample pretreatment step 10, potentially enables achievinga detection limit down to a few microbial cells in the sample.

Performing an assay based on rRNA, as opposed to gDNA, as the primarymode of detection, may provide enhanced sensitivity for two reasons.Firstly, rRNA is not as stable as gDNA and thus foreign rRNA has a muchsmaller chance of being introduced into the thermal chamber as a majorcontamination component. Secondly, the number of rRNA molecules becomingavailable after electrical treatment of step 11, for even a single cell,is in the range of 10⁴, which, according to the methods disclosedherein, will generally exceed the quantity of contaminant backgroundmolecules.

Following the completion of step 13, the liquid contains sufficient cDNAand/or amplicons to be split into multiple aliquots and delivered into aplurality of wells, in a manner such that each well receives astatistically significant number of target molecules.

Test Panels for Rapid De-Escalation of Antimicrobial Therapy Based onProcessing of Direct Samples

In some embodiments, the panel of rRNA tests may be selected to enablerapid de-escalation of antibiotic therapy from one or morebroad-spectrum antibiotics to one or more narrow-spectrum antibiotics.Using narrow-spectrum antimicrobial drugs not only provides a benefit interms of cost reduction, but it also limits the potential for adverseeffects such as superinfection and the development of drug-resistantmicrobes. As noted above, the present embodiments enable the rapiddetection and/or identification of infectious agents, which couldimprove the decision-making process of health care professionals, andhelp them to administer narrow spectrum antimicrobial drugs at anearlier time. This may prevent the development of resistance, reducetoxicity, and substantially reduce healthcare costs.

Unlike known molecular methods that are focused on the targetedidentification of an organism at the species level based on acomprehensive, species-level test panel, such as the Septifast system,selected embodiments described herein provide sufficient information forrapid de-escalation of antimicrobial therapy, without needing to providefull species and/or genus level identification for every expectedpathogen. The reduced panels described herein are suitable for effectiverapid re-vectoring of antimicrobial treatment because many organismswill respond to a common narrow-spectrum antibiotic, and as such, thetest panel need not identify each and every genus and/or species of theexpected pathogens, an instead need only identify those organisms orgroups of organisms whose presence or absence will impact antimicrobialtherapy and guide the selection of appropriate narrow-spectrumantibiotics. In many of the embodiments provided herein, a test panel isselected to provide information that is not exclusive to the genus orspecies level, but is instead includes a combination of rRNA testresults at the kingdom, Gram-status, genus, and species levels.

Accordingly, in some embodiments, a test panel for rapid de-escalationof antimicrobial therapy includes, at a minimum, the following:

(a) one or more primary rRNA tests at the kingdom level to determinewhether the organism is a bacterial or a fungal organism;

(b) one or more secondary tests to identify the Gram status of theorganism, if bacterial; and

(c) one or more tertiary rRNA tests, for each of fungi, Gram-positivebacteria, and Gram-negative bacteria, to identify at least one genus orspecies of the organism, where the result of the tertiary test issuitable for selecting an appropriate narrow-spectrum antibiotic.

In some embodiments, one or more of the rRNA tertiary tests may bereplaced and/or augmented by a strain level test, where the strain leveltest may be a gDNA test suitable for identification of a selectedstrain.

It has been found that such test panels are sufficient for rapidde-escalation of antimicrobial therapy from broad spectrum antibioticsto narrow spectrum antibiotics, especially when the test is performed ona direct, non-enriched sample, prior to enrichment or culturing.

The selection of one or more tertiary tests at the species or genuslevel will depend on a number of factors, such as the nature of theantibiogram, and the availability of suitable antibiotics. Non-limitingexamples of test panels are described below. For example, over time, asuitable genus or species level tertiary test for Gram-negative bacteriamay change due to changes in pathogen prevalence, changes in antibioticresistance, and/or the availability of new antibiotics.

In some embodiments, the reduced test panel is selected by mapping theavailable antibiotics to information associated with the highest levelsof pathogenic taxa, and the test panel is selected to include the testsfor these highest levels. For example, the test panel may include, at ahigh level, three multiplex tests for isolating a true positive sampleto one of Gram-positive, Gram negative, or fungi. This may be achieved,as noted above, by isolating specific regions of the ribosome belongingto all Gram-positive bacteria, to the exclusion of all others, forexample, it becomes possible to identify a true Gram-positive sample. Ifthis same procedure is repeated for all Gram-negative bacteria to theexclusion of all others, it becomes possible to identify a trueGram-negative sample. A third test would isolate and identify a truefungal sample.

This reduction principle may be repeated in a hierarchical format (forexample, a decision tree or a flow chart) to determine the set of testsneeded to de-escalate antimicrobial therapy. This effectively reducesthe very large number of tests for all possible organisms to a subset oftests relevant to antimicrobial re-vectoring.

For example, a suitable test panel may involve a kingdom-based primarytest for bacterial vs. fungal organisms, a Gram-status secondary test,and a tertiary test for the genus of Streptococci, and an additionalspecies-level tertiary test, provided that the species-level test wouldenable further antimicrobial de-escalation. For example, a species-leveltertiary test for Streptococcus pneumoniae would enable furtherde-escalation, based on presently available antibiotics.

In another example, a suitable tertiary test may be for the genus ofStaphylococcus, and the test panel may include an additionalspecies-level tertiary test for the species of Staphylococcus aureus. Asample testing positive to both of these tertiary tests would be thoughtto include Staphylococcus aureus, and a medical practitioner couldsubsequently decide to de-escalate antibiotic therapy to Vancomyacin andOxacillin due to the awareness of potential methicillin resistance. Onthe other hand, if the sample found to be positive to Gram-positive andStaphylococcus, but negative to S. aureus, then the medical practitionermay determine that the offending organism is probably Staphylococciother than S. aureus and most probably a contaminant, which could leadthe medical practitioner to determine that treatment is not warranted.

This principle is illustrated in FIGS. 9(a) and 9(b). In FIG. 9(a), ahierarchical panel diagram is shown in which the number of tests thatdetermines a course of antimicrobial therapy for blood infections hasbeen reduced to nine. FIG. 9(b) shows an example of organismidentification based on different possible panel test results, alongwith a corresponding determination of a suitable antibiotic. Such apanel is important in at least two respects. Firstly, the number oftests is significantly reduced, compared to species-level test panels.For example, there are well over 100 possible sources of bacterial orfungal infections, and testing for each and every one would be anarduous and complicated task, making a rapid diagnosis and targetedtreatment difficult. Secondly, the degree of confidence with eachhierarchical level in the panel tree is increased, which could verypossibly impact treatment decisions on a real-time basis. This approach,in effect, has a built-in control that improves the confidence interval.

Three additional example panels are presented in FIGS. 10(a) through(c). The panel shown in FIG. 10(a) provides the following information:an indication of the kingdom classification (bacterial or fungal) of thepathogen, the Gram status of a bacteria, and further identification ofselected genus and/or species levels. As noted above, identification ofpathogens at a family and/or genus level may assist in decision makingfor antimicrobial drug selection and further testing as necessary. Inthis example panel, identification of selected pathogens at a specieslevel provides the physician with specific information that is relevantto de-escalation of antimicrobial treatment, and information that canwarrant further testing. For example, the identification of S. aureus atthe species level could indicate to the physician that further testingfor MRSA is warranted. Also, for example, the identification of S.pneumoniae at species level could alarm the physician to increasingampicillin-resistant strains of S. pneumoniae. Furthermore, theidentification of P. aeruginosa at species level could support adecision to alter the antimicrobial therapy to a more targeted,narrow-spectrum antimicrobial drug that is more selective for P.aeruginosa. Similarly, identification of Aspergillus can assistdecision-making for more appropriate and narrow-spectrum treatment,while other fungi are generally sensitive to wider spectrum antimycoticagents.

In the panel shown in FIG. 10(b), pathogen information is provided atthe family, genus and/or species level in a more selective manner thatis correlated with the selection of narrow-spectrum and appropriatelytargeted antimicrobial therapy. This panel is formulated based on theassessment that other than S. aureus, similar antibiotics can beconsidered for other Gram-positive bacteria. As a result, the S. aureusspecies level test is sufficient to provide suitable information forde-escalation of antibiotic therapy if the pathogen is found to beGram-positive. Furthermore, if S. aureus is identified, the physicianmay request a test known for the mecA gene in order to determine whetheror not the pathogen is MRSA. As noted above, the detection andidentification for the panels shown in FIGS. 10(a) and 10(b) may beperformed by detecting specific nucleic acid sequences within thepathogen's rRNA.

In the example panel shown in FIG. 10(c), the mecA gene a strain leveltest is provided for the detection of MRSA, which renders the specieslevel test for S. aureus unnecessary. The test for the mecA gene may beperformed based on the detection of genomic DNA. If the pathogen isfound to be Gram-positive but not MRSA, then suitable other antibioticswhich can cover all Gram-positive pathogens can be considered fortreatment.

The aforementioned rapid sample pretreatment and rRNA-based testingprotocols, when combined with a rapid de-escalation test panel asdescribed above, enables the rapid and effective selection ofappropriate narrow-spectrum antibiotics, or the appropriate initialselection of a suitable antibiotic, thereby dramatically reducing thenumber of tests, and the complexity of medical inquiry, required toenable a therapeutic decision. In particular, when aforementionedmethods of sample pretreatment and rapid rRNA reverse transcription PCRare employed to perform tests according to such a rapid de-escalationpanel, results are provided on a suitable timescale to affect patientoutcomes, and with sufficient information to guide the de-escalation ofantimicrobial therapy.

As noted above, the present embodiments do not require an enrichmentstep, and are capable of rapidly providing results that influenceantimicrobial treatment. For example, in some embodiments, the timedelay between initiating the pretreatment phase and the availability ofthe test results may be less than approximately 30 minutes. The rapidavailability of the test results is important for enabling de-escalationof antimicrobial treatment, or prescribing an initial narrow-spectrumantibiotic, on a clinically relevant and effective timescale.

This is to be contrasted with existing molecular methods, which fail tofind widespread clinical utility due to (1) the requirement for complexand time consuming manual sample preparation steps, (2) insufficientrecovery of microbial cells when performing sample preparation based ondirect, non-enriched samples, and (3) overly complex test results thatfail to clearly inform appropriate decision making in antimicrobialstewardship.

The following examples are presented to enable those skilled in the artto understand and to practice embodiments of the present disclosure.They should not be considered as a limitation on the scope of thepresent embodiments, but merely as being illustrative and representativethereof.

EXAMPLES

In the following examples, Gram-negative bacteria cells were grown on LBagar plates and a single colony of cells was cultured in LB brothovernight at 37° C. Gram-positive bacterial and fungal cells were grownon tryptic soy agar with 5% sheep blood and a single colony of cells wascultured in tryptic soy broth overnight at 37° C. The cells werecentrifuged at 7000 rpm for 5 min. The cell pellet was washed twice andre-suspended in 0.8 mM phosphate buffer pH 7.4, pre-filtered through a0.2 μm filter.

The blood cell lysis reagent was used whenever blood sample pretreatmentwas required. The blood cell lysis reagent consisted of a mixture ofsaponin (84510, Sigma), sodium polyanethol sulfanate (SPS) (P2008,Sigma) and poly(propylene glycol) (PPG) MW 2000 (202339, Sigma). Saponinwas dissolved in reagent grade water, filtered through 0.2 μm PESsyringe filter and purified using Amicon Ultra-15 10K MW cut-off(Z706345, Sigma). SPS was dissolved in reagent water and filteredthrough 0.2 μm PES syringe filter. PPG MW 2000 (202339, Sigma) was useddirectly from the original bottle. In addition, Fluorinert™ FC-40(F9755, Sigma) was added to serve as cushioning liquid.

Unless any variation is specified, the following example pretreatmentprocedure which includes blood cell lysis step followed by 4 wash cycleswas performed in experiments which required pretreatment of bloodsamples. 10 μL of Fluorinert™ was added to 2 mL siliconizedmicrocentrifuge tubes (T3531, Sigma), followed by the addition of 500 μLof the blood cell lysis reagent. The blood cell lysis reagent consistedof 75 mg/mL saponin, 15 mg/mL SPS and 1% PPG. Sodium citrate-treatedhuman whole blood (Bioreclemation Inc.) of 1 mL spiked with microbialcells was added to the tube containing the blood cell lysis reagent andthe Fluorinert™, and mixed by inverting ten times and vortexing at lowspeeds for 10 sec. The final concentrations of the components in themixture were 25 mg/mL saponin, 5 mg/mL SPS and 0.33% PPG. The tubes werecentrifuged at 12,000 rpm for 1 min and 1.35 mL of the supernatant wasremoved, leaving 150 μL of the liquid supernatant, Fluorinert™ and thesedimented microbial I cells. The first wash cycles was performed byadding 1.35 mL of 0.8 mM phosphate buffer to the remaining liquidsupernatant of 150 μL as described above, mixing by vortexing at lowspeed for 10 sec, centrifugation at 12,000 rpm for 1 min and removal of1.35 mL of the supernatant, leaving 150 μL of the liquid supernatant,the Fluorinert™ and the sedimented microbial. The remaining wash cycleswere performed by adding 0.75 mL of 0.8 mM phosphate buffer to theremaining liquid supernatant of 150 μL, mixing by vortexing at low speedfor 10 sec, centrifugation at 12,000 rpm for 1 min and removal of 0.75mL of the supernatant, leaving 150 μL of the liquid supernatant, theFluorinert™ and the sedimented microbial. After the last wash, thesedimented microbial cells were re-suspended in the residual liquid,whose volume was in the range of 100 to 300 ul. Positive control samplesare prepared by spiking 0.8 mM phosphate buffer pH 7.4 with the sameconcentration of respective microbial cells as the nominal concentrationof the pretreated sample in respective experiments.

In examples that employed electrical cell lysis, the pretreated samplesand the positive control samples were passed through an electricalchamber with steps of 10 μL/10 s and applying n=250 bipolar squarepulses having duration of 50 μs and amplitude of 190 V. The electricalchamber had a dimension of 6.4×15×0.2 mm³ and the outlet and outletports were of restricted type.

In the following examples, real-time reverse transcription PCR(real-time RT-PCR) assay was performed to detect a specific targetregion in 16S or 18S rRNA of respective microbial cell types. Theprimers were designed by sequence alignment software (Bioedit, IbisBiosciences, USA) and primer design software (Primer3, NationalInstitutes of Health). The cell lysate of the pretreated sample and thespiking control following the electrical lysis was subjected toreal-time RT-PCR. In addition to the samples, the following negativecontrols were subjected to real-time RT-PCR: pre-filtered phosphatebuffer used for cell suspension (negative control; buffer), and unspikedblood subjected to the same pretreatment and electrical lysis protocolas the spiked blood samples (negative control; blood).

Unless any variation was specified, RT-PCR reaction of 20 μL volume wasprepared by mixing 5 μL of sample, 10 μL of Kapa 2G Robust Hotstart2×PCR reaction mix (kk5515, KAPA Biosystems), 1.2 μL of reversetranscriptase (GoScript, A5004 Promega), 1 μL of forward primer (10 μM),1 μL of reverse primer (10 μM), 1 μl of SYTO-9 (100 nM, S34854,Invitrogen) and 0.8 μL of RNAase free water. One-step real time RT-PCRwas performed by reverse transcription at 55° C. for 5 min, inactivationof reverse transcription at 95° C. for 2 min, followed by 35 cycles ofcDNA amplification (denaturation at 95° C. for 3 sec, annealing atrespective temperature for 3 sec, and extension at 72° C. for 3 sec) inEco Real Time PCR system (illumina) using a double stranded DNA bindingfluorescent dye, SYTO-9.

Example 1: The Effect of Washing on the Reduction of the QuenchingEffect of Lysed Blood Debris on Amplicon Detection by Fluorescent Signal

The experiments presented in this example were performed to determinethe effect of washing cycles of reducing the quenching effect of lysedblood debris on the fluorescent signal and enabling efficient real timeRT-PCR assay, according to an example method of pretreatment andamplification.

A suspension of 2000 CFU/mL Pseudomonas aeruginosa cells in 0.8 mMphosphate buffer with pH 7.4 was electrically lysed. The lysate wassubjected to real time RT-PCR to prepare sufficient volume of ampliconsfor the experiment in this example. The forward and reverse primers usedfor RT-PCR were GGGCAGTAAGTTAATACCTTGC (SEQ. ID. 25) andTCTACGCATTTCACCGCTACAC (SEQ. ID. 26), respectively. The 16S rRNA genefragment of 251 bases pair at a conserved region of P. aeruginosa(nucleotides 438 to 689 using P. aeruginosa D77P as a reference) wasamplified. Human whole blood, which had been drawn into a collectiontube with sodium citrate anticoagulant were subjected to the examplesample pretreatment procedure described above with four wash cycles asdescribed above (involving centrifugation). At the end of each washcycle the removed supernatant was stored. A 5 μL volume of amplicon,prepared as described above, was added to 5 μL of the stored supernatantof each wash cycle. The fluorescence signal was read in Illuminamachine. The signals were normalized to the signal of amlicons added tothe clean phosphate buffer. The results of three different runs werepresented in FIG. 11(a). As it is observed, the fluorescence quenchingeffects of the debris are significant up to 3 times washing, whichcorresponds to a 500 fold dilution.

Example 2: the Effect of Dilution-Based Wash Steps and Microbial CellLysis Method on Molecular Assays

The experiments presented in this example were performed to demonstratehow number of wash steps and microbial cell lysis method affect theinhibitory effects of the lysed blood debris on the downstream molecularassays.

Pseudomonas aeruginosa cells were used and the cell suspension wasprepared as described above. Human whole blood was subjected to thesample pretreatment procedure as described above with the followingexceptions. The wash cycles were performed by adding the respectivevolume of 0.8 mM phosphate buffer to the remaining liquid supernatant of150 uL and mixing by inverting 10 times, without vortexing orcentrifugation. Thus, the samples are simply the serial dilutions of theoriginally lysed blood. The dilution-based wash cycles were performedfive times instead of four times as per the example sample pretreatmentprocedure described above that employed centrifugation when washing. Theremoved supernatant of each wash cycle was split into 3 aliquots andspiked with P. aeruginosa cells. Each aliquot of 75 μL included nominal30 microbial cells. One aliquot was electrically lysed and one aliquotwas heat lysed by heating the suspension at 95° C. for 10 minutes. Thethird aliquot was subjected to mechanical lysis using glass beads (GB).To perform GB lysis, an equal volume of glass beads (<106 μm, G4649Sigma) was added to 75 μL of cell suspension and mechanically lysed byvortexing at a high speed for 2 minutes. The GB cell lysate wascentrifuged at 14,000 rpm for 1 minute to separate the beads and thesupernatant was collected for the assay.

All lysates of 5 μL each containing the equivalent of 2 lysed microbialcells were assayed with real-time RT-PCR alongside negative control(buffer) and positive control (buffer spiked with microbial cells andelectrically lysed). The forward and reverse primers used for RT-PCRwere GGGCAGTAAGTTAATACCTTGC (SEQ. ID. 25) and TCTACGCATTTCACCGCTACAC(SEQ. ID. 26), respectively. The 16S rRNA gene fragment of 251 basespair at a conserved region of P. aeruginosa (nucleotides 438 to 689using P. aeruginosa D77P as a reference) was amplified. The resultingsignal versus real time PCR cycle # plots are presented in FIG. 11(b)for lysates of the fourth wash cycle and FIG. 11(c) for lysates of thefifth wash cycle. As it is observed in FIG. 11 (b), electrical and heatlysis greatly reduce the inhibitory effects of blood debris present inthe supernatant of fourth wash cycle. However, as it is observed in FIG.11 (c), in the absence of centrifugation, a dilution equivalent to fivewash cycles is required to eliminate the inhibitory effect of blooddebris on the performance of RT-PCR assay when using the reagents andconditions similar to the present experiments.

Example 3: the Effect of Washing During the Example Sample PretreatmentProcedure on Reducing Inhibitory Effects of Lysed Blood Debris onMolecular Assays

The experiments presented in this example were performed to demonstratehow washing cycles and electrical treatment reduces the inhibitoryeffects of the lysed blood debris on the downstream molecular assays.The dependence of a typical RT-PCR assay on the number of washing cyclesduring the example sample pretreatment was tested.

Pseudomonas aeruginosa cells were used and the cell suspension wasprepared as described above.

Human whole blood was subjected to the example sample pretreatmentprocedure with four wash cycles as described above. The removedsupernatants of third and fourth wash cycles were spiked with P.aeruginosa cells and subjected to electrical lysis. Each wash cyclesupernatant aliquot of 75 μL included nominal 30 microbial cells. Alllysates of 5 μL each containing the equivalent of 2 lysed microbialcells were assayed with real-time RT-PCR alongside positive control(buffer spiked with microbial cells and electrically lysed). The forwardand reverse primers used for RT-PCR were GGGCAGTAAGTTAATACCTTGC (SEQ.ID. 25) and TCTACGCATTTCACCGCTACAC (SEQ. ID. 26), respectively. The 16SrRNA gene fragment of 251 bases pair at a conserved region of P.aeruginosa (nucleotides 438 to 689 using P. aeruginosa D77P as areference) was amplified.

The resulting signal versus cycle # plots is presented in FIG. 11(d). Asit is observed, by following the example sample pretreatment wash stepsdescribed above, electrical treatment greatly reduces the inhibitoryeffects of blood debris. While the Ct value in the case of GB lysis wasabove 35 cycles, the corresponding Ct for electrical lysis was 25cycles. In other words, the effects of electrical treatment in reducingthe inhibitory effects of blood lysis debris, is equivalent to dilutinginhibitors by a factor of 13. Moreover, in the case of the RT-PCRreagents used in the present tests, 4 washes are sufficient foreliminating the inhibitory effects of blood debris.

Example 4: Dependence of Detection Efficiency on the Blood Lysis ReagentComposition and Pretreatment Vessel Interior Surface

The experiments presented in the following examples were performed todemonstrate the efficiency by which the microbial cells in a whole bloodsample are recovered into the pretreated sample and that molecular assayinhibitory compounds are removed or inactivated. As noted above, suchmethods may be applicable for downstream reagentless microbial celllysis followed by a nucleic acid assay without requiring additionalsteps of nucleic acid extraction/purification and/or medium balance. Thedependence of microbial cell recovery on blood cell lysis reagentcomposition, volume of cushioning liquid and interior surface propertyof the sample pretreatment vessel were verified.

In these experiments, the viability of the recovered microbial cells maynot be of concern, which is in contrast to cases when microbial cellsare intended for subsequent growth (such as in the disclosures of Dornreferenced above). Instead, it may be sufficient that the blood celllysis reagent does not substantially damage the microbial cells, to theextent of releasing nucleic acids and other macromolecules of interest,and avoids the introduction of nucleic acid assay inhibitory agents inthe pretreated sample. Accordingly, the blood cell lysis reagentcomposition may be selected such that it is appropriate for wide rangeof microorganisms, in particular Gram-negative bacteria that are lesstolerant of strong blood cell lysis agents. In order to demonstrate thecapability of the method for the preparation of such less tolerantbacteria, the following examples were performed with Pseudomonasaeruginosa as the test organism.

P. aeruginosa cells were cultured and the cell suspension was preparedas described above. 10 μL of Fluorinert™ was added to 2 mL siliconizedmicrocentrifuge tubes, followed by the addition of 500 μL of the bloodcell lysis reagent. Despite any variation is specified, human wholeblood collected in BD vacutainer with sodium citrate (BD366415) is used.The blood spiked with P. aeruginosa in 1 mL volume at 600 CFU/mL wasadded to the tube containing the blood cell lysis reagent and theFluorinert™ and mixed by inverting 10 times and vortexing at low speedsfor 10 sec. The sample pretreatment was performed as described above.After the last wash, most of the supernatant was removed and thesedimented cells were re-suspended in 300 μL of 0.8 mM phosphate bufferpH 7.4. The resulting pretreated sample contained a nominal cell numberof 600. A 60 μL volume of the pretreated samples and the positivecontrol were lysed by electrical lysis as described above. 5 μL of thecell lysate, containing the equivalent of 10 lysed microbial cells, andthe negative control were assayed with real-time RT-PCR. The forward andreverse primers used for RT-PCR were GGGCAGTAAGTTAATACCTTGC (SEQ. ID.25) and TCTACGCATTTCACCGCTACAC (SEQ. ID. 26), respectively. The 16S rRNAgene fragment of 251 bases pair at a conserved region of P. aeruginosa(nucleotides 438 to 689 using Pseudomonas aeruginosa D77P as areference) was amplified.

Blood cell lysis reagent with varying concentrations of saponin and SPS,and varying volumes of Fluorinert™ were tested to verify thesignificance of each component in the pretreatment mixture. Then, usingthe optimized composition of the pretreatment mixture, bacteria recoveryfrom pretreatment vessels with or without interior surface treatment wasinvestigated.

Example 4.1: Variation in Concentrations of Saponin Concentrations onReal-Time RT-PCR Assay Signal

Human whole blood of 1 mL, spiked to 600 CFU/mL P. aeruginosa was addedto 0.5 mL of the pretreatment mixture containing 10 μL of Fluorinert™FC-40, 4.5 to 240 mg/mL saponin, 15 mg/mL SPS and 1% PPG, in a 2 mLsiliconized microcentrifuge tube. The final concentrations of thecomponents in the mixture were 1.5 to 80 mg/mL saponin, 5 mg/mL SPS and0.33% PPG. The sample pretreatment procedure was performed and thepretreated samples and positive controls were electrically lysed. Thereal time RT-PCR assay signal of the bacteria in the pretreated samplerecovered from the whole blood after the sample pretreatment ispresented in FIG. 12(a). It appears that a final concentration ofsaponin of approximately 25 mg/mL is the most effective concentrationfor the present experimental conditions.

Example 4.2: Variation in Concentrations of SPS on Real-Time RT-PCRAssay Signal

Human whole blood of 1 mL, spiked at 600 CFU/mL P. aeruginosa was addedto 0.5 mL of the pretreatment mixture containing 10 μL of Fluorinert™FC-40, 75 mg/mL saponin, SPS in the range of 0.5-20 mg/mL and 1% PPG, in2 mL siliconized microcentrifuge tube. The final concentrations of thecomponents in the mixture were 25 mg/mL saponin, 0.5 to 20 mg/mL SPS and0.33% PPG The real time RT-PCR assay signal of the bacteria in thepretreated sample recovered from the whole blood after the samplepretreatment is presented in FIG. 12 (b). It appears that a finalconcentration of SPS of approximately 5 mg/mL is the most effectiveconcentration for the present experimental conditions.

Example 4.3: Variation in Type of Anticoagulant Used for Blood and thePretreatment Mixture Composition Adjustment on Real-Time RT-PCR AssaySignal

Human whole blood, which had been drawn into BD Vacutainer collectiontubes with different anticoagulants. The collection tubes used weresupplied with buffered sodium citrate (BD366415, normally used forcoagulation testing), K₂ EDTA (BD367863, normally used for hematologytesting), sodium heparin (BD367871, normally used for chemistry testing)and SPS containing 1.7 mL of 0.35% SPS and 0.85% sodium chloride(BD364960, specialty tubes normally used for blood culture specimencollection). The standard volume of blood specified by the manufacturerwas drawn into each tube. Human whole blood collected in citrate, EDTAand Heparin tubes of 1 mL each, spiked at 600 CFU/mL P. aeruginosa wasadded to 0.5 mL of the pretreatment mixture containing 10 μL ofFluorinert™ FC-40, 75 mg/mL saponin, 15 mg/mL SPS and 1% PPG, in 2 mL(T-3531, Sigma) siliconized microcentrifuge tubes. The finalconcentrations of the components in the mixture were 25 mg/mL saponin, 5mg/mL SPS and 0.33% PPG. For the blood collected in SPS tube, due to thepresence of additional SPS in the anticoagulant solution in the tube,the pretreatment mixture was adjusted as 0.5 mL of the mixturecontaining 10 μL of Fluorinert™ FC-40, 112.5 mg/mL saponin, 7.5 mg/mLSPS and 1% PPG.

The pretreatment procedure was performed and the samples wereelectrically lysed. The real time RT-PCR assay signal of the bacteria inthe pretreated sample recovered from the whole blood after the samplepretreatment is presented in FIG. 13. It was demonstrated that microbialcells were recovered from human whole blood despite the difference inthe types of coagulants used.

Example 4.4: Variation in Volumes of Fluorinert™ on Real-Time RT-PCRAssay Signal

Human whole blood of 1 mL, spiked at 600 CFU/mL P. aeruginosa was addedto 0.5 mL of the pretreatment mixture containing different volumes ofFluorinert™ FC-40, 75 mg/mL saponin, 15 mg/mL SPS and 1% PPG. Inaddition the effect of variation in Fluorinert™ volume was tested in 2different siliconized microcentrifuge tubes with different tube bottomgeometry; 2 mL tubes (T-3531, Sigma) and 1.7 mL tubes (T-3406, Sigma).The final concentrations of the components in the mixture were 25 mg/mLsaponin, 5 mg/mL SPS and 0.33% PPG. The pretreatment procedure wasperformed and the samples were electrically lysed. The real time RT-PCRassay signal of the bacteria in the pretreated sample recovered from thewhole blood after the sample pretreatment is presented in FIGS. 14(a)and (b), where (a) shows the assay signal for the 2 ml tubes, and (b)shows the assay signal for the 1.7 ml tubes. It was demonstrated thatfor the present experimental condition, the volume of Fluorinert™ shouldbe more than approximately 2.5 μL and less than approximately 100 μL forboth types of tubes.

Example 4.5: Variation in Pretreatment Vessel Interior Surface Propertyon Real-Time RT-PCR Assay Signal

Human whole blood of 1 mL, spiked at 600 CFU/mL P. aeruginosa was addedto 0.5 mL of the pretreatment mixture containing 10 μL of Fluorinert™FC-40, 75 mg/mL saponin, 15 mg/mL SPS and 1% PPG, in 2 mLmicrocentrifuge tube with or without interior surface treatment. In onegroup, regular 2 mL polypropylene microcentrifuge tubes (B71420,Bioplastics) were used. In the other group, 2 mL siliconizedmicrocentrifuge tubes (T-3531, Sigma) were used. The finalconcentrations of the components in the mixture were 25 mg/mL saponin, 5mg/mL SPS and 0.33% PPG. The real time RT-PCR assay signal versus cyclenumber of the bacteria in the pretreated sample recovered from wholeblood after sample pretreatment is presented in FIG. 15. The C_(T) valuefor regular tube is about 4 cycles later than the case of siliconizedtube, indicating more efficient cell recovery during the pretreatmentprocess. Therefore, the siliconization of the interior surface may beemployed for more efficient recovery of bacterial cells from the wholeblood.

Example 4.6: Variation in Type of Blood Cell Lysing Reagent on Real-TimeRT-PCR Assay Signal

Human whole blood of 1 mL, spiked at 600 CFU/mL P. aeruginosa was addedto the pretreatment mixture containing 10 μL of Fluorinert™ FC-40 in a 2mL microcentrifuge tube. The pretreatment reagent used in this examplewere (a) 500 μL of saponin mixture containing 75 mg/mL saponin, 15 mg/mLSPS and 1% PPG and (b) 1 mL of 1% Triton X-100 in Sodium Carbonatebuffer pH 10. After the centrifugation, following the cell lysis step,the supernatant was removed, leaving 150 μL of the liquid supernatant,Fluorinert™ and the sedimented microbial cells. Five washing cycles ofpretreatment procedure were performed and the samples were electricallylysed. The real time RT-PCR assay signal of the bacteria in the treatedsample recovered from the whole blood indicated that the Ct values ofthe two blood cell lysing reagents formulations were similar within 1cycle.

Example 4.7: Variation in Type of Blood Cell Lysing Reagent on Real-TimeRT-PCR Assay Signal

Human whole blood of 1 mL, spiked at 600 CFU/mL P. aeruginosa was addedto the pretreatment mixture containing 10 μL of Fluorinert™ FC-40 in a 2mL microcentrifuge tube. The pretreatment reagent of 500 μL used in thisexample were (a) saponin mixture containing 75 mg/mL saponin, 15 mg/mLSPS and 1% PPG, (b) 1.5% Triton X-100 and 18.75 mg/mL SPS in SodiumPhosphate buffer pH 7.4, (c) 1.5% Triton X-100 and 22.5 mg/mL SPS inSodium Phosphate buffer pH 7.4, (d) 4.5% Triton X-100 and 15 mg/mL SPSin Sodium Phosphate buffer pH 7.4 and (e) 3% Triton X-100 and 22.5 mg/mLSPS in Sodium Phosphate buffer pH 7.4. After the centrifugation,following the cell lysis step, the supernatant was removed, leaving 150μL of the liquid supernatant, Fluorinert™ and the sedimented microbialcells. Five washing cycles of the pretreatment procedure were performedand the samples were electrically lysed. The real time RT-PCR assaysignal of the bacteria in the treated sample recovered from the wholeblood indicated that the Ct values of the blood cell lysing reagentswere similar within 1 to 2 cycles.

Example 5: Detection Limit of Microorganisms Spiked in Human Whole Blood

The three examples presented in this section demonstrate the ability ofthe device to detect microbial cells in human whole blood with adetection limit in the range of 1-10 cells per mL for fungi,Gram-positive bacteria and Gram-negative bacteria.

Human whole blood (Bioreclemation Inc.) of 1 mL volume was spiked withthe microbial cells specific to each example below and subjected topretreatment and electrical lysis as described above. As a positivecontrol, the same concentration of the microbial cells in 0.8 mMphosphate buffer was subjected to electrical lysis without pretreatment.

Human whole blood of 1 mL spiked with 300 and 30 CFU/mL of microbialcells was added to the tube containing blood cell lysis reagent andFluorinert™, and the example sample pretreatment procedure describedabove was performed.

After the last wash, the supernatant was removed and the sedimentedcells were re-suspended in 300 μL of 0.8 mM phosphate buffer pH 7.4(resulting in a nominal concentration of 10³ and 10² CFU/mL).

A 60 μL volume of the pretreated samples and the positive controls weresubjected to electrical lysis. In each case, a cell lysate volume of 10μL (nominally 10 and 1 cells respectively per sample) was subjected toamplification by RT-PCR.

In the following examples, RT-PCR reaction mixture of 25 μL volume wasprepared by mixing 10 μL of sample, 12.5 μL of 2×PCR reaction mix (2GRobust HotStart, KAPA Biosystems), 1.3 μL of reverse transcriptase(GoScript, Promega), 0.6 μl of forward primer (10 μM) and 0.6 μL ofreverse primer (10 μM). One-step real time RT-PCR was performed byreverse transcription at 55° C. for 5 min, inactivation of reversetranscription at 95° C. for 2 min, followed by 35 cycles of cDNAamplification (denaturation at 95° C. for 3 sec, annealing at 55° C. for3 sec, and extension at 72° C. for 3 sec) and final extension at 72° C.for 1 min in Eco Real-Time PCR System (illumina). The resulting RT-PCRproduct of 15 μL was resolved by gel electrophoresis on 1% agarose gelin 0.5×TBE buffer and 0.5 μg/mL ethidium bromide at 150 volts for 30min. The specific nucleotide sequences within the amplified region weredetected using specific molecular beacon. RT-PCR product of 5 μL wasmixed with 1 μL of the buffer containing 20 mM Tris-HCl pH 8, 10 mM KCland 7 mM MgCl₂ as well as 1 μM of molecular beacon. The mixture washeated at 95° C. for 30 sec to denature the amplicon and the molecularbeacon, followed by cooling to room temperature, allowing hybridizationof the molecular beacon to the target sequence. The resulting mixture of6 μL was transferred to a micro-well and the fluorescence intensity ofexcitation at 492 nm and emission at 517 nm was measured usingfluorescence microscopy (LumaScope, etaluma).

Example 5.1: Detection of Fungi Spiked in Human Whole Blood

Samples spiked with C. albicans were prepared and processed as describedabove for RT-PCR assay. The one-step RT-PCR protocol used UFF3 forwardprimer (5′-AACGAAAGTTAGGGGATCGAAG-3′) (SEQ. ID. 15) and UFR3 reverseprimer (5′-CTTTAAGTTTCAGCCTTGCGA-3′) (SEQ. ID. 16). The 18S rRNA genefragment of 167 bases pair at a hypervariable region of all fungispecies (nucleotides 940 to 1107 using Candida albicans AB013586 as areference) was amplified.

The resulting RT-PCR product of 15 μL was resolved by gelelectrophoresis and the amplified region of 18S rRNA was observed inFIG. 16. This example demonstrates the detection of C. albicans fungalcells spiked in human whole blood with sensitivity in the range of 1-6cells.

The specific nucleotide sequences within the amplified region weredetected using the molecular beacon6-FAM-5′-CCGAGCCGTAGTCTTAACCATAAACTATGCGCT-3′-DABCYL (SEQ. ID. 17)(nucleotides 977 to 997 using Candida albicans AB013586 as a reference),designed to detect all fungal pathogens. The detection signals of thepositive controls and the spiked blood samples recovered from bloodpretreatment are presented in FIG. 17. This example demonstrates theefficiency of molecular beacon method to detect C. albicans spiked inhuman whole blood with sensitivity in the range of 1-6 cells.

Example 5.2: Detection of Gram-Positive Bacteria Spiked in Human WholeBlood

Samples spiked with S. pneumoniae were prepared and processed asdescribed above for RT-PCR assay. The one-step RT-PCR protocol used Ubeaprimer pair (UbeaF forward primer; 5′-GACAGGTGGTGCATGGTTGTC-3′ (SEQ. ID.18) and UbeaR reverse primer, 5′-ACGTCATCCCCACCTTCCTC-3′) (SEQ. ID. 19)and GP1 primer pair (GP1F forward primer; 5′-ATGCATAGCCGACCTGAGAG-3′(SEQ. ID. 11) and GP1R reverse primer; 5′-AGTTAGCCGTSSCTTTCTG-3′) (SEQ.ID. 12). The 16S rRNA gene fragment of 170 bases pair at a hypervariableregion of all bacterial species (nucleotides 1035 to 1185 usingStreptococcus pneumonia G54 as a reference) was amplified by universalbacterial Ubea primer pair and the 16S rRNA gene fragment of 230 basepairs at a hypervariable region of all Gram-positive bacterial species(nucleotide 290 to 520 using Staphylococcus aureus NR 037007 asreference) was amplified by Gram-positive specific GP1 primer pair.

The resulting amplified product using GP1 primer pair, targeting allGram-positive bacterial species was resolved by gel electrophoresis andthe amplified fragment of 16S rRNA was observed in FIG. 18. This exampledemonstrates the detection of Gram-positive S. pneumoniae bacteria inblood with sensitivity in the range of 1-6 cells.

The Gram-positive specific nucleotide sequences within the amplifiedproduct using Ubea universal bacterial primer pair, targeting allbacterial species were detected using the molecular beacon;6-FAM-5′-CCGAGCTTAGTTGCCATCATTTAGTTGGGCACTCTAGCTCGG-3′-DABCYL (SEQ. ID.20) (nucleotides 1113 to 1142 using Streptococcus pneumonia G54 as areference) designed to detect all Gram-positive bacterial pathogens. Thedetection signals of the positive controls and the spiked blood samplesrecovered from blood pretreatment were presented in FIG. 19. Thisexample demonstrates the efficiency of molecular beacon method to detectGram-positive S. pneumoniae spiked in human whole blood with sensitivityin the range of 1-6 cells.

Example 5.3: Detection of Gram-Negative Bacteria Spiked in Human WholeBlood

Samples spiked with E. coli were prepared and processed as describedabove for RT-PCR assay. The one-step RT-PCR protocol used UB primer pair(UBF8 forward primer; 5′-CGGCTAACTCCGTGCCAGCAG-3′ (SEQ. ID. 12) and UBRsreverse primer; 5′-ATCTCTACGCATTTCACCGCTACAC-3′) (SEQ. ID. 2) and GN1primer pair (GN1F forward primer; 5′-GTTACCCGCAGAAGAAGCAC-3′ (SEQ. ID.13) and GN1R reverse primer; 5′-ACCTGAGCGTCAGTCTTCGT-3′ (SEQ. ID. 14)targeting all Gram-negative bacterial species. The 16S rRNA genefragment of 203 base pairs at a hypervariable region of all bacterialspecies (nucleotides 504 to 707 using Escherichia coli O104:H4 str.2011C-3493 as a reference) was amplified by universal bacterial UBprimer pair and the 16S rRNA gene fragment of 278 bases pair in ahypervariable region of all Gram-negative bacterial species (nucleotide475 to 753 using Escherichia coli NR 024570.1 as reference) wasamplified by Gram-negative specific GN1 primer pair.

The resulting amplified product using GN1 primer pair, targeting allGram-negative bacterial species was resolved by gel electrophoresis andthe amplified fragment of 16S rRNA was observed in FIG. 20. This exampledemonstrates the detection of Gram-negative E. coli bacteria in bloodwith sensitivity in the range of 1-6 cells.

The Gram-positive specific nucleotide sequences within the amplifiedproduct using UB universal bacterial primer pair, targeting allbacterial species were detected using the molecular beacon;6-FAM-5′-CCGAGCGGTGCAAGCGTTAATCGGAATTACTGGGCGCTCGG-3′-DABCYL (SEQ. ID.5) (nucleotides 541-569 using Escherichia coli O104:H4 str. 2011C-3493as a reference) designed to detect all Gram-negative bacterialpathogens. The detection signals of the spiking controls and the spikedblood samples recovered from blood pretreatment were presented in FIG.21. This example demonstrates the efficiency of molecular beacon methodto detect Gram negative E. coli spiked in human whole blood withsensitivity in the range of 1-6 cells.

Example 6: Repeatability of Detection Method as Measured Using Real-TimeRT-PCR

This example demonstrates the repeatability of the detection ofmicrobial cells in 1 mL of human whole blood sample within a totalrun-time of 30 minutes. Although the end goal is a multiplexed detectionscheme, the present example is the detection of each species byindividual RT-PCR assays.

Five different species each of Gram-positive, Gram-negative and fungalmicrobial cells described in FIG. 22, the representatives of the examplepanels in FIGS. 9 and 10, were tested. Sixty cells of each species werespiked into 1 mL of blood. The blood samples were then pre-treated toobtain 300 μL of pretreated samples in filter-sterilized 0.8 mMphosphate buffer and designated as spiked blood samples. Sixty cells ofeach species were added into 300 μL of filter-sterilized 0.8 mMphosphate buffer and used as respective positive controls. The blood of1 mL volume without spiking with microbial cells also was pre-treated toobtain 300 μL of a pretreated sample in filter-sterilized 0.8 mMphosphate buffer and designated as a negative control (blood). Eachsample and control of 70 μL was electrically lysed and RT-PCR assay wasperformed on 5.5 μL of each lysate, which is equivalent to detection ata single cell level. RT-PCR reaction of 20 μL volume was prepared bymixing 5.5 μL of sample, 10 μl of 2×PCR reaction mix (GoTaq Colourless,Promega), 1.5 μL of reverse transcriptase (GoScript, Promega), 0.5 μl offorward primer (10 μM), 0.5 μl of reverse primer (10 μM) and 2 μl ofSYTO-9 (100 nM). One-step real-time RT-PCR was performed by reversetranscription at 55° C. for 5 min, inactivation of reverse transcriptionat 95° C. for 2 min, followed by 30 cycles of cDNA amplification at 95°C. for 3 sec, 59° C. for 3 sec, and 72° C. for 3 sec in Eco real timePCR system (Illumina) using a double stranded DNA binding fluorescentdye, SYTO-9. The primers used for RT-PCR to detect Gram-positivebacteria, Gram-negative bacteria and fungi are presented in FIG. 23.

The duration of sample pretreatment process was 6 minutes. Theelectrical lysis of samples or controls in steps of 10 μL/10 s tookplace in 1 minute. The RT-PCR assay with real time detection was under20 minutes. Therefore, the total run-time for the detection of microbialcells in 1 mL of raw human whole blood was 27 minutes.

The detectability criterion was defined as the following. The RT-PCRfluorescence signal versus cycle number for the case of a singleEnterococcus faecalis cell in human whole blood is presented in FIG. 24.The standard deviation of the signal over the first 10 cycles, where thesignal is predominately background noise, is calculated and indicated asa large dashed line in FIG. 24. A threshold signal level is decided tobe 6σ. The cycle number where the recorded signal exceeds the thresholdlevel is defined as C_(T). If the C_(T) value of a sample differs bymore than 5 cycles (a signal to noise ratio of over 32) from the C_(T)value of the corresponding negative control blood sample running inparallel with the sample, the detection is considered to be unambiguous.

Example 6.1: Rapid Detection of Gram-Positive Bacteria in Raw HumanWhole Blood

Five pathogenic species were tested: EF (Enterococcus faecalis), SS(Streptococcus sanguis), SP (Streptococcus pneumonia), SA(Staphylococcus aureus), and SPy (Streptococcus pyogenes). Spiked bloodsamples, and positive and negative controls were prepared and subjectedto electrical lysis as described above. The RT-PCR protocol used GPF8forward primer; 5′-GCTCGTGTCGTGAGATGTTGGG-3′ (SEQ. ID. 21) and GPR8reverse primer, 5′-CAGGTCATAAGGGGCATGATGAT-3′ (SEQ. ID. 22). The 16SrRNA gene fragment of 151 base pairs at a hypervariable region of allGram-positive bacterial species (nucleotides 1069 to 1220 usingStaphylococcus sp. LS255 as a reference) was amplified.

The resulting C_(T) values for different species over five independentruns are presented in FIG. 25. The detection limit criterion asdescribed above was satisfied for all Gram-positive species.

Example 6.2: Rapid Detection of Gram-Negative Bacteria in Raw HumanWhole Blood

Five pathogenic species were tested: KP (Klebsiella pneumoniae), PA(Pseudomonas aeruginosa), EC (Escherichia coli), ECL (Enterobactercloacae), and AB (Acinetobacter baumannii). Spiked blood samples, andpositive and negative controls were prepared and subjected to electricallysis as described above. The RT-PCR protocol used GNF8 forward primer;5′-GTTACCCGCAGAAGAAGCACCG-3′ (SEQ. ID. 23) and GNR8 reverse primer,5′-ATGCAGTTCCCAGGTTGAGCC-3′ (SEQ. ID. 24). The 16S rRNA gene fragment of151 base pairs at a hypervariable region of all Gram-negative bacterialspecies (nucleotides 484 to 635 using Escherichia coli IaI1 as areference) was amplified. The resulting C_(T) values for differentspecies over five independent runs are presented in FIG. 26. Thedetection limit criterion as described above was satisfied for allGram-negative species.

Example 6.3: Rapid Detection of Fungi in Raw Human Whole Blood

Five pathogenic species were tested: CA (Candida albicans), CrN(Cryptococcus neoformans), CG (Candida glabrata), CK (Candida krusei,and AF (Aspergillus fumigatus). Spiked blood samples, and positive andnegative controls were prepared and subjected to electrical lysis asdescribed above. The RT-PCR protocol used UFF2 forward primer;5′-ACGGGGAGGTAGTGACAATAAAT-3′ (SEQ. ID. 9) and UFR2 reverse primer,5′-CCCAAGGTTCAACTACGAGCTT-3′ (SEQ. ID. 10). The 18S rRNA gene fragmentof 190 base pairs at a hypervariable region of all fungal species(nucleotide 434 to 624 using Candida albicans AB013586 as a reference)was amplified. The resulting C_(T) values for different species overfive independent runs are presented in FIG. 27. The detection limitcriterion as described above was satisfied for all fungal species.

The specific embodiments described above have been shown by way ofexample, and it should be understood that these embodiments may besusceptible to various modifications and alternative forms. It should befurther understood that the claims are not intended to be limited to theparticular forms disclosed, but rather to cover all modifications,equivalents, and alternatives falling within the spirit and scope ofthis disclosure.

Therefore what is claimed is:
 1. A method of extracting microbial cellsfrom a blood containing sample, the method comprising: adding a bloodcontaining sample to a pretreatment vessel, the pretreatment vesselcomprising a pretreatment mixture, the pretreatment mixture comprising ablood cell lysis reagent and a hydrophobic cushioning liquid having adensity greater than that of the blood containing sample and the bloodcell lysis reagent; agitating the contents of the pretreatment vessel;centrifuging the pretreatment vessel such that the cushioning liquidforms a liquid interface below a supernatant, wherein microbial cellsfrom the blood containing sample are removed from suspension andcollected adjacent to the liquid interface; withdrawing a substantialquantity of the supernatant without removing the collected microbialcells; agitating the contents of the pretreatment vessel and allowingthe cushioning liquid to re-establish the liquid interface below asuspension containing the microbial cells; and extracting a least aportion of the suspension without extracting a substantial portion ofthe cushioning liquid, thereby obtaining an extracted suspensioncomprising microbial cells.
 2. The method according to claim 1 whereinthe extracted suspension is substantially free of the cushioning liquid.3. The method according to claim 1 wherein prior to agitating thecontents of the pretreatment vessel and allowing the cushioning liquidto reestablish the liquid interface below the suspension containing themicrobial cells, the following washing steps are performed: adding avolume of wash buffer to the pretreatment vessel; agitating the contentsof the pretreatment vessel; centrifuging the pretreatment vessel suchthat the cushioning liquid forms a liquid interface below a supernatant,and such that microbial cells are transported to and collected at theliquid interface; and withdrawing a substantial quantity of thesupernatant without extracting the collected microbial cells.
 4. Themethod according to claim 3 further comprising performing the washingsteps one or more additional times.
 5. The method according to claim 1wherein after centrifugation, the liquid interface is formed on a sideportion of the pretreatment vessel, wherein the method further comprisesre-orienting the pretreatment vessel such that the liquid interface issubstantially horizontal, and wherein reorientation of the pretreatmentvessel is performed such that a substantial quantity of the microbialcells remain collected at the liquid interface.
 6. The method accordingto claim 1 wherein the volume of the cushioning liquid is betweenapproximately 2.5 microliters and 100 microliters.
 7. The methodaccording to claim 1 wherein the volume of the cushioning liquid isbetween approximately 5 microliters and 15 microliters.
 8. The methodaccording to claim 1 wherein the volume of the cushioning liquid isbetween approximately 0.1% and 10% of the volume of the pretreatmentvessel.
 9. The method according to claim 1 wherein the volume ofcushioning liquid is provided such that the surface of the pretreatmentvessel that is wetted by the cushioning liquid envelops at least theregion of the pretreatment vessel surface where the microbial cellswould sediment under centrifugation in the absence of the cushioningliquid.
 10. The method according to claim 1 wherein the blood cell lysisreagent is an aqueous solution containing saponin in an amount rangingbetween approximately 1.5 and 80 mg/ml, and sodium polyanetholesulfonatein an amount ranging between approximately 0.5 and 20 mg/ml, expressedin terms of the mixture of blood containing sample and blood cell lysisreagent.
 11. The method according to claim 10 wherein the blood celllysis reagent further comprises an antifoaming agent.
 12. The methodaccording to claim 11 wherein the antifoaming agent is poly(propyleneglycol).
 13. The method according to claim 1 wherein the blood celllysis reagent is an aqueous solution containing saponin in an amountranging between approximately 10 and 30 mg/ml, and sodiumpolyanetholesulfonate in an amount ranging between approximately 2.5 and10 mg/ml, expressed in terms of the mixture of blood containing sampleand blood cell lysis reagent.
 14. The method according to claim 1wherein the blood cell lysis reagent is an aqueous solution containingpolyethylene glycol tea-octylphenyl ether in an amount ranging betweenapproximately 0.05 and 2.5%, and sodium polyanetholesulfonate in anamount ranging between approximately 5 and 10 mg/ml, expressed in termsof the mixture of blood containing sample and blood cell lysis reagent.15. The method according to claim 14 wherein the pH of the blood celllysis reagent is between approximately 5 and
 11. 16. The methodaccording to claim 14 wherein the pH of the blood cell lysis reagent isbetween approximately 9 and
 11. 17. The method according to claim 14wherein polyethylene glycol tert-octylphenyl ether is provided in anamount ranging between approximately 0.5 and 1.5%.
 18. The methodaccording to claim 1 further comprising: lysing the microbial cellswithin the extracted suspension to obtain a lysate; and performing atleast one assay to detect at least one analyte that may be present inthe lysate.
 19. The method according to claim 18 wherein the step oflysing the microbial cells is performed electrically.
 20. The methodaccording to claim 19 wherein the electrical lysis is performedaccording to the steps of: providing a microfluidic device comprising: afluidic channel having an upper channel surface, a lower channelsurface, a side wall, and a thickness on a submillimeter scale; an upperelectrode on the upper channel surface; and a lower electrode on thelower channel surface; wherein the channel is adapted to support flashheating of the extracted suspension under the application of voltagepulses to the electrodes; flowing the extracted suspension into thechannel; and applying a series of bipolar voltage pulses between theupper electrode and the lower electrode, wherein an amplitude, pulsewidth, and duration of the voltage pulses are sufficient to flash heatthe extracted suspension and lyse the microbial cells, thereby releasingmacromolecules from the microbial cells.
 21. The method according toclaim 20 wherein the voltage pulses are applied such that Joule heatingof the extracted suspension occurs with a rate of at least approximately250 degrees per second.
 22. The method according to claim 20 whereinunder application of the voltage pulses, the extracted suspension isheated faster than a timescale of thermal diffusion from the channel.23. The method according to claim 20 wherein thermal properties of thechannel surfaces are selected to such that, in response to appliedvoltage pulses, Joule heating of the extracted suspension occurs with arate of at least approximately 2000 degrees per second.
 24. The methodaccording to claim 18 wherein at least one of the assays is performed bypolymerase chain reaction.
 25. The method according to claim 18 whereinat least one of the assays is performed, at least in part, by reversetranscription polymerase chain reaction.
 26. The method according toclaim 18 wherein at least one of the analytes is rRNA.
 27. The methodaccording to claim 1 wherein the microbial cells are bacterial and/orfungal cells.